Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Ciliate diversity and distribution across an environmental and depth gradient in Long Island Sound,USA

The nature and extent of microbial biodiversity remain controversial with persistent debates over patterns of distributions (i.e. cosmopolitanism versus endemism) and the processes that structure these patterns (neutrality versus selection). We used culture‐independent approaches to address these issues focusing on two groups of ciliates, the Oligotrichia (Spirotrichea) and Choreotrichia (Spirotrichea) across an environmental gradient. We assessed SSU rDNA diversity in ciliate communities at six stations in Long Island Sound spanning the frontal region that separates the fresher Connecticut River outflow plume from the open Sound. As in previous studies, we find one abundant cosmopolitan species (Strombidium biarmatum), a few moderately abundant sequences, and a long list of rare sequences. Furthermore, neither ciliate diversity nor species composition showed any clear relationship to measured environmental parameters (temperature, salinity, accessory pigment composition and chorophyll). Overall, we observed that diversity decreased moving from nearshore to offshore. We also conducted analyses to detect clustering among the sampled communities using the software Unifrac. This approach revealed three significant clusters grouping samples from nearshore, surface and deep/well mixed stations. We find no strong fit of our communities to log series, geometric or log normal distributions, though one of the 3 clusters is most consistent with a log series distribution. However, when we remove the abundant cosmopolitan species S. biarmatum, all three clusters fit to a log series distribution. These analyses suggest that, with the exception of one cosmopolitan species, the oligotrich and choreotrich communities at these stations may be distributed in a neutral manner.     

Developments in the taxonomy of silica‐scaled chrysophytes – from morphological and ultrastructural to molecular approaches

Taxonomy in silica‐scaled chrysophytes has gone through three morphological phases. From primary studies of the cell morphology in the 18th century, the focus was in the 20th century replaced by studies of the silica structures of the cell envelope. Now, in the latest decades the importance of DNA sequencing has been recognized, not only to support the taxonomic framework but also to obtain new understanding of taxonomic relations among particular taxa. In the first part of this review, we provide a historical overview of the developments in the taxonomy of scale‐bearing chrysophytes. In the second part, we present a phylogenetic reconstruction of chrysophyte algae, updated by newly obtained SSU rDNA and rbcL sequences of several isolated Synura, Mallomonas and Chrysosphaerella species. We detected significant incongruence between the phylogenies obtained from the different datasets, with the SSU rDNA phylogram being the most congruent with the morphological data. Significant saturation of the first rbcL codon position could indicate the presence of positive selection in the rbcL dataset. Within the Synurales, the relationships revealed by the phylogenetic analyses highlight the artificial infragenetic classification of Mallomonas and Synura, and the occurrence of cryptic diversity within a number of traditionally defined species. Finally, three new combinations are proposed based on the phylogenetic analyses: Tessellaria lapponica, Synura asmundiae and S. bjoerkii.     

Molecular Phylogeny of Marine Gregarine Parasites (Apicomplexa) from Tube‐forming Polychaetes (Sabellariidae,Cirratulidae, and Serpulidae), Including Descriptions of Two New Species of Selenidium

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube‐forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single‐cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube‐forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

Halosarpheia japonica sp. nov. (Halosphaeriales, Ascomycota) from marine habitats in Japan

A new Halosarpheia species, collected from driftwood from Hakkeijima beach, Yokohama, Japan, is described and illustrated and is compared with other species of the genus. The new fungus was growing together with its anamorph on a piece of decaying wood. SSU and LSU rDNA sequences for both morphs were 99% similar. Phylogenetic analyses of SSU and LSU rDNA sequences of the both morphs confirm their anamorph–teleomorph relationship and placed the new fungus in the Halosarpheia sensu stricto clade with high statistical support. Halosarpheia japonica is characterized by its polar appendage that is initially enclosed in a cellular sheath and dissolves in water, the appendage then swells to form a huge tree-like structure. The other three species currently included in Halosarpheia sensu stricto differ from H. japonica by having two polar appendages that uncoil in water to form long filaments.     

Taxonomic Descriptions of One New Species and Redescription of Four Species in the Family Strombidiidae (Ciliophora,Oligotrichida)1

ABSTRACT Five ciliate species collected from the Woods Hole area were examined by protargol silver impregnation and scanning electron microscopy. These ciliates have been shown to sequester and use chloroplasts obtained from flagellate prey. One new species, Strombidium chlorophilum, is described. Four other species, Strombidium capitatum (Leegaard, 1915) Kahl, 1932, Strombidium conicum (Lohmann, 1908) Wulff, 1919, Strombidium acutum (Leegaard, 1915) Kahl, 1932, and Laboea strobila Lohmann, 1908, are redescribed. Characters used in describing the Strombidiidae include cell size and shape, anterior and ventral polykinetids, macronuclear shape and size, the kinetid “girdle,” the ventral kinety, the trichites, and the paroral kinety. The rationale for using these characters as taxonomic criteria is discussed.     

Gyrodinium jinhaense n. sp., a New Heterotrophic Unarmored Dinoflagellate from the Coastal Waters of Korea

Four unarmored heterotrophic dinoflagellates were isolated from the coastal waters of southern Korea. The rDNA sequences of four clonal cultures were determined, and the morphology of one of the four strains was examined using light and scanning and transmission electron microscopy. The large subunit (LSU) and small subunit (SSU) rDNA sequences of each of the strains differed by 0–0.9% from those of the other strains, and the SSU rDNA sequence of the strain differed by 1.8–4.4% from those of other Gyrodinium species, whereas the LSU (D1–D2) rDNA sequence differed by 12.4–22.2%. Furthermore, phylogenetic trees showed that Gyrodinium jinhaense n. sp. formed a distinctive clade among the other Gyrodinium species. Meanwhile, microscopy revealed an elliptical bisected apical structure complex and a cingulum that was displaced by approximately one‐quarter of the cell length, which confirmed that the dinoflagellate belonged to the genus Gyrodinium. However, the cell surface was ornamented with 16 longitudinal striations, both on the episome and hyposome, unlike other Gyrodinium species. Furthermore, the cells were observed to have pusule systems and trichocysts but lacked mucocysts. Based on morphology and molecular data, we consider this strain to be a new species in the genus Gyrodinium and thus, propose that it be assigned to the name G. jinhaense n. sp.     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Phylogenetic Reappraisal of the Genus Monomorphina (Euglenophyceae) Based on Molecular and Morphological Data

A morphological and molecular examination of the genus Monomorphina was conducted on 46 strains isolated mainly from Korea. The strains were divided into two types based on morphological data: Monomorphina aenigmatica and M. pyrum ‐ like species. Phylogenetic analysis based on a combined data set of nuclear SSU and LSU and plastid SSU and LSU rDNA showed that the strains could be divided into eight clades: Clade A of M. aenigmatica, Clade B of the isolates (M. pyropsis) from Michigan, USA, Clade C of M. pseudopyrum, Clade D of the isolates (M. pyroria) from Bremen, Germany, Clade E of M. soropyrum, Clade F of M. pyriformis, Clade G of M. parapyrum, and Clade H of M. pyrum. Six of these clades came from strains that would be considered M. pyrum sensu Kosmala et Zakry?, one of which could be recognized as a traditional species (M. pyrum) and five were designated as new species; each species had unique molecular signatures at nr SSU rDNA helix 17 and 17′ and spacer E23_14′‐E23_15. The species of Monomorphina had a wide range of genetic diversity with interspecies sequence similarity of 85.6%–97.1% and intraspecies similarity of 96.4%–99.9%. Our results suggested that genetic diversity found in the M. pyrum complex justifies the recognition of a minimum of eight species within this genus, based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

INTRAGENOMIC NUCLEOTIDE POLYMORPHISM AMONG SMALL SUBUNIT (18S) RDNA PARALOGS IN THE DIATOM GENUS SKELETONEMA (BACILLARIOPHYTA)1

Morphological features of the siliceous cell wall traditionally have been used to diagnose and classify species of diatoms, though an increasing number of studies distinguish new species, in part, by phylogenetic analysis of rDNA sequences. Intragenomic sequence variation is common among the hundreds to thousands of rDNA cistrons present within a genome, and this variation has strong potential to obscure species boundaries based on rDNA sequences. We screened six Skeletonema culture strains for intragenomic nucleotide polymorphisms in the small subunit (SSU) rDNA gene and found that all strains had polymorphic sites, with proportions ranging from 0.57% to 1.81%. In all cases, transitions accounted for more than 70% of nucleotide differences at polymorphic sites. Polymorphic sites were split nearly evenly in the SSU rRNA molecule between the base‐paired regions of helices (52%) and the unpaired regions of loops and bulges (48%). Phylogenetic analysis showed that SSU rDNA genotypes were monophyletic for two of the six culture strains examined. Genotypes from the other four culture strains either showed little or no phylogenetic structure compared with genotypes of other conspecific culture strains or had phylogenetic structure that was incongruent with existing species boundaries. Moderate to strong support for monophyly was recovered for four of the seven species included in the analysis. Phylogenetic results combined with the low sequence divergence of SSU rDNA genotypes within species suggest that concerted evolution has not proceeded to completion in these species and/or that the rate at which variation is being generated exceeds the rate at which concerted evolution is expunging variation.     

Molecular phylogeny of hipposiderid bats (Chiroptera: Hipposideridae) from Solomon Islands and Cape York Peninsula,Australia

Throughout the Old World Tropics, examples of cryptic diversity have been demonstrated in leaf‐nosed bats from the Family Hipposideridae. In addition to cryptic diversity, the widespread distributions and graded morphologies of some species render the taxonomy of this family poorly resolved. We used sequences of the mitochondrial gene ND2 and the nuclear gene RAG1 to reconstruct molecular phylogenetic relationships for hipposiderid species. Specimens from Solomon Island and Cape York Peninsula, Australia, were sequenced and combined with published sequences from Southeast Asia. Our results suggest the presence of undescribed diversity in northern Australia. Sequences of ND2 and RAG1 for samples of Cape York Hipposideros ater were distinct from those of Southeast Asia, with corrected sequence divergence estimates for the mitochondrial gene exceeding 23%. In comparison, there was no cryptic diversity detected in Solomon Island specimens when compared to conspecifics from Southeast Asia. On the contrary, three species members of the diadema morphological group from the region exhibit variability in the mitochondrial gene ND2, far below that of other species included in the analyses.     

Molecular investigation of Phryganella acropodia Hertwig et Lesser, 1874 (Arcellinida,Amoebozoa)

Phryganella acropodia Hertwig and Lesser, 1874, is one of the most common and abundant testate amoeba species. It represents the type species of the genus Phryganella Penard, 1902, which in turn is the type genus for the suborder Phryganellina (Arcellinida) Bovee, 1985, but despite its taxonomic importance it was not yet analyzed with molecular methods. We established two cultures of putative Phryganella acropodia, designed Phryganellina-specific primers, amplified SSU rDNA data and subjected these sequences to phylogenetic analyses. Morphological and genetic differences were found between both strains. With SSU rDNA phylogenetic analyses we confirm that Phryganella acropodia branches with Phryganella paradoxa Penard, 1902 and Cryptodifflugia Penard, 1890 in the Phryganellina. We thus give further evidence that pseudopodia morphology in the Arcellinida is a character of high taxonomic value, as suggested by Bovee and Jung when erecting the suborder Phryganellina. Moreover, we provide evidence for cryptic diversity and for the first time confirm the existence of a naked life stage in Arcellinida by molecular means.     

Multiple transisthmian divergences,extensive cryptic diversity,occasional long‐distance dispersal,and biogeographic patterns in a marine coastal isopod with an amphi‐American distribution

Excirolana braziliensis is a coastal intertidal isopod with a broad distribution spanning the Atlantic and Pacific tropical and temperate coasts of the American continent. Two separate regional studies (one in Panama and one in Chile) revealed the presence of highly genetically divergent lineages, implying that this taxon constitutes a cryptic species complex. The relationships among the lineages found in these two different regions and in the rest of the distribution, however, remain unknown. To better understand the phylogeographic patterns of E. braziliensis, we conducted phylogenetic analyses of specimens from much of its entire range. We obtained DNA sequences for fragments of four mitochondrial genes (16S rDNA, 12S rDNA, COI, and Cytb) and also used publicly available sequences. We conducted maximum likelihood and Bayesian phylogenetic reconstruction methods. Phylogeographic patterns revealed the following: (1) new highly divergent lineages of E. braziliensis; (2) three instances of Atlantic–Pacific divergences, some of which appear to predate the closure of the Isthmus of Panama; (3) the distributional limit of highly divergent lineages found in Brazil coincides with the boundary between two major marine coastal provinces; (4) evidence of recent long‐distance dispersal in the Caribbean; and (5) populations in the Gulf of California have closer affinities with lineages further south in the Pacific, which contrasts with the closer affinity with the Caribbean reported for other intertidal organisms. The high levels of cryptic diversity detected also bring about challenges for the conservation of this isopod and its fragile environment, the sandy shores. Our findings underscore the importance of comprehensive geographic sampling for phylogeographic and taxonomical studies of broadly distributed putative species harboring extensive cryptic diversity.