A survey of Polytomella (Chlorophyceae,Chlorophyta) strains in public culture collections

Polytomella is a genus of colorless green algae in the Reinhardtinia clade of the Chlamydomonadales, which has proven useful for a broad range of studies particularly those exploring the evolutionary loss of photosynthesis and mitochondrial genomics/biochemistry. Although 13 Polytomella strain accessions are currently available from public culture collections, the taxonomic status and redundancy of many of these strains is not clear because of possible mix‐ups, deficient historical records, and incomplete molecular data. This study therefore considers previously available and/or new cox1 and mitochondrial DNA telomere sequences from all 13 Polytomella strain accessions. Among four of these, namely P. parva SAG 63‐3, P. piriformis SAG 63‐10, P. capuana SAG 63‐5, and P. magna SAG 63‐9, cox1 and mitochondrial telomere regions are both highly divergent between strains. All of the remaining nine Polytomella strain accessions have cox1 sequences that are identical to that of P. parva SAG 63‐3 and although five of these have a mitochondrial telomere haplotype that is identical to that of P. parva SAG 63‐3, the remaining four have one of three different haplotypes. Among the 10 strains with identical cox1 sequences, we suggest that three of the telomere haplotypes are associated with distinct geographical isolates of Polytomella and the fourth evolved from one of these isolates during 50 years of active culture.     

The mitochondrial ATP synthase of chlorophycean algae contains eight subunits of unknown origin involved in the formation of an atypical stator-stalk and in the dimerization of the complex

Mitochondrial F₁F _O -ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator-stalk as well as those involved in the dimerization of the fungal and mammal complex. Instead, it contains eight novel polypeptides named ASA1 to 8. We show that homologs of these subunits are also present in the chlorophycean algae Polytomella sp. and Volvox carterii. Blue Native Gel Electrophoresis analysis of mitochondria from different green algal species also indicates that stable dimeric mitochondrial ATP synthases may be characteristic of all Chlorophyceae. One additional subunit, ASA9, was identified in the purified mitochondrial ATP synthase of Polytomella sp. The dissociation profile of the Polytomella enzyme at high-temperatures and cross-linking experiments finally suggest that some of the ASA polypeptides constitute a stator-stalk with a unique architecture, while others may be involved in the formation of a highly-stable dimeric complex. The algal enzyme seems to have modified the structural features of its surrounding scaffold, while conserving almost intact the structure of its catalytic subunits.     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

Resolving the phylogenetic relationship between Chlamydomonas sp. UWO 241 and Chlamydomonas raudensis sag 49.72 (Chlorophyceae) with nuclear and plastid DNA sequences

The Antarctic psychrophilic green alga Chlamy‐domonas sp. UWO 241 is an emerging model for studying microbial adaptation to polar environments. However, little is known about its evolutionary history and its phylogenetic relationship with other chlamydomonadalean algae is equivocal. Here, we attempt to clarify the phylogenetic position of UWO 241, specifically with respect to Chlamydomonas rau‐densis SAG 49.72. Contrary to a previous report, we show that UWO 241 is a distinct species from SAG 49.72. Our phylogenetic analyses of nuclear and plastid DNA sequences reveal that UWO 241 represents a unique lineage within the Moewusinia clade (sensu Nakada) of the Chlamydomonadales (Chlorophyceae, Chlorophyta), closely affiliated to the marine species Chlamydomonas parkeae SAG 24.89.     

Conjugation in Polytomella agilis Araga˜o (Volvocida,Polyblepharidae)*

SYNOPSIS. The occurrence of conjugation in the flagellate, Polytomella agilis Aragão, as first reported in 1910, is confirmed. The gametes made contact with each other in a variety of positions. Our observations differ in minor ways from those reported for P. agilis and are in considerable disagreement with others published on sexual reproduction in Polytomella caeca.     

EVOLUTIONARY ORIGIN OF GLOEOMONAS (VOLVOCALES,CHLOROPHYCEAE), BASED ON ULTRASTRUCTURE OF CHLOROPLASTS AND MOLECULAR PHYLOGENY1

Gloeomonas is a peculiar unicellular volvocalean genus because it lacks pyrenoids in the chloroplasts under the light microscope and has two flagellar bases that are remote from each other. However, ultrastructural features of chloroplasts are very limited, and no molecular phylogenetic analyses have been carried out in Gloeomonas. In this study, we observed ultrastructural features of chloroplasts of three species of Gloeomonas and Chloromonas rubrifilum (Korshikov ex Pascher) Pröschold, B. Marin, U. Schlösser et Melkonian SAG 3.85, and phylogenetic analyses were carried out based on the combined data set from 18S rRNA, ATP synthase beta‐subunit, and P700 chl a–apoprotein A2 gene sequences to deduce the natural phylogenetic positions of the genus Gloeomonas. The present EM demonstrated that the chloroplasts of the three Gloeomonas species and C. rubrifilum SAG 3.85 did not have typical pyrenoids with associated starch grains, but they possessed pyrenoid matrices that protruded interiorly within the stroma regions of the chloroplast. The pyrenoid matrices were large and broad in C. rubrifilum, whereas those of the three Gloeomonas species were recognized in only the small protruded regions of the chloroplast lobes. The present multigene phylogenetic analyses resolved that the three species of Gloeomonas belong to the Chloromonas lineage or Chloromonadinia of the Volvocales, and Chloromonas insignis (Anakhin) Gerloff et H. Ettl NIES‐447 and C. rubrifilum SAG 3.85, both of which have pyrenoids without associated starch grains, were positioned basally to the clade composed of the three species of Gloeomonas. Therefore, Gloeomonas might have evolved from such a Chloromonas species through reduction in pyrenoid matrix size within the chloroplast and by separating their two flagellar bases.     

Another Origin of Coloniality in Volvocaleans: The Phylogenetic Position of Pyrobotrys Arnoldi (Spondylomoraceae,Volvocales)

ABSTRACT. Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a‐apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic “Pascherina tetras” SAG 159‐1 was also analyzed.     

Antibody Detection and Molecular Characterization of Toxoplasma gondii from Bobcats (Lynx rufus), Domestic Cats (Felis catus), and Wildlife from Minnesota,USA

Little is known of the epidemiology of toxoplasmosis in Minnesota. Here, we evaluated Toxoplasma gondii infection in 50 wild bobcats (Lynx rufus) and 75 other animals on/near 10 cattle farms. Antibodies to T. gondii were assayed in serum samples or tissue fluids by the modified agglutination test (MAT, cut‐off 1:25). Twenty nine of 50 bobcats and 15 of 41 wildlife trapped on the vicinity of 10 farms and nine of 16 adult domestic cats (Felis catus) and six of 14 domestic dogs resident on farms were seropositive. Toxoplasma gondii oocysts were not found in feces of any felid. Tissues of all seropositive wild animals trapped on the farm were bioassayed in mice and viable T. gondii was isolated from two badgers (Taxidea taxus), two raccoons (Procyon lotor), one coyote (Canis latrans), and one opossum (Didelphis virginiana). All six T. gondii isolates were further propagated in cell culture. Multi‐locus PCR‐RFLP genotyping using 10 markers (SAG1, SAG2 (5′‐3′SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22‐8, c29‐2, L358, PK1, and Apico), and DNA from cell culture derived tachyzoites revealed three genotypes; #5 ToxoDataBase (1 coyote, 1 raccoon), #1 (1 badger, 1 raccoon, 1 opossum), and #2 (1 badger). This is the first report of T. gondii prevalence in domestic cats and in bobcats from Minnesota, and the first isolation of viable T. gondii from badger.     

Mechanistic differences in the uptake of salicylic acid glucose conjugates by vacuolar membrane‐enriched vesicles isolated from Arabidopsis thaliana

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2‐O‐β‐d ‐glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane‐enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A₁ (vacuolar H⁺‐ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H⁺‐antiporter. Despite its absence in the vacuole, we observed the MgATP‐dependent uptake of SGE by Arabidopsis vacuolar membrane‐enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A₁ and gramicidin D providing evidence that uptake was dependent on an H⁺‐antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP‐dependent SAG and SGE uptake exhibited Michaelis–Menten‐type saturation kinetics. The vacuolar enriched‐membrane vesicles had a 46‐fold greater affinity and a 10‐fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long‐term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations.     

Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii

Aim: Development of a ‘miniprimer’ PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart’s bacterial wilt on maize. Methods and Results: Four 10‐nucleotide (10‐nt) ‘miniprimer’ sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni‐BacF‐10/Uni‐BacR‐10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 ‘clone types’ identified, sequences of a 1·23‐kb fragment had a 99·8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon ( AY166713 ). Other dominant cloned fragments included a 411‐bp amplicon that exhibited 99·8% similarity to the psaU gene (syn:ysaU; GQ249669 ), a type III protein‐secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548‐bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99. Conclusion: The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii. Significance and Impact of the study: This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart’s wilt pathogen of maize.     

Mitogenomic phylogeny of Acanthocephala reveals novel Class relationships

The Acanthocephala is a phylum of obligate endoparasitic animals comprising four classes (Archiacanthocephala, Palaeacanthocephala, Eoacanthocephala and Polyacanthocephala), although the phylogenetic interrelationships of these classes still remains unresolved. To investigate phylogenetic relationships of major acanthocephalan groups, we characterized the complete mitochondrial genome sequences of two palaeacanthocephalan species Centrorhynchus aluconis and Prosthorhynchus transversus (representing two different families of the order Polymorphida), and Polyacanthorhynchus caballeroi (the first mitogenomic representative of the class Polyacanthocephala) and used these new sequences for phylogenetic analyses, along with 32 platyzoan mtDNAs, including 10 additional acanthocephalans. Phylogenetic analyses using concatenated amino acid sequences for 12 protein‐coding genes with maximum likelihood and Bayesian inference methods supported monophyly of Acanthocephala. Within the phylum, Archiacanthocephala was positioned as the sister to the clade containing all three other acanthocephalan classes, with the polyacanthocephalan species P. caballeroi nested within Eoacanthocephala. This result contradicts morphology‐based classification systems that treated polyacanthorhynchids as one of the palaeacanthocephalan families, and instead suggests Polyacanthocephala is a member of Eoacanthocephala. Within the Palaeacanthocephala, Polymorphida monophyly was strongly supported and this is inconsistent with nuclear rDNA‐based molecular hypotheses that suggest non‐monophyly.     

Phylogenetic positioning of the Antarctic alga Prasiola crispa (Trebouxiophyceae) using organellar genomes and their structural analysis

Antarctica is one of the most difficult habitats for sustaining life on earth; organisms that live there have developed different strategies for survival. Among these organisms is the green alga Prasiola crispa, belonging to the class Trebouxiophyceae. The literature on P. crispa taxonomy is scarce, and many gaps in the evolutionary relationship with its closest relatives remain. The goal of this study was to analyze the evolutionary relationships between P. crispa and other green algae using plastid and mitochondrial genomes. In addition, we analyzed the synteny conservation of these genomes of P. crispa with those of closely related species. Based on the plastid genome, P. crispa grouped with Prasiolopsis sp. SAG 84.81, another Trebouxiophyceaen species from the Prasiola clade. Based on the mitochondrial genome analysis, P. crispa grouped with other Trebouxiophyceaen species but had a basal position. The structure of the P. crispa chloroplast genome had low synteny with Prasiolopsis sp. SAG 84.81, despite some conserved gene blocks. The same was observed in the mitochondrial genome compared with Coccomyxa subellipsoidea C‐169. We were able to establish the phylogenetic position of P. crispa with other species of Trebouxiophyceae using its genomes. In addition, we described the plasticity of these genomes using a structural analysis. The plastid and mitochondrial genomes of P. crispa will be useful for further genetic studies, phylogenetic analysis and resource protection of P. crispa as well as for further phylogenetic analysis of Trebouxiophyceaen green algae.     

A new type of 3‐D peripheral ultrastructure in Glaucocystis (Glaucocystales,Glaucophyta) as revealed by ultra‐high voltage electron microscopy

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three‐dimensional (3‐D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra‐high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3‐D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3‐D ultrastructure resolved in “G. incrassata” SAG 229‐2 cells by 3‐D modeling based on UHVEM tomography using high‐pressure freezing and freeze‐substitution fixation. The plasma membrane and underlying flattened vesicles in “G. incrassata” SAG 229‐2 exhibited grooves at intervals of 200–600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3‐D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3‐D ultrastructure of the protoplast periphery is discussed.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

Nucleotide Diversity of the Colorless Green Alga Polytomella parva (Chlorophyceae,Chlorophyta): High for the Mitochondrial Telomeres,Surprisingly Low Everywhere Else*

ABSTRACT. Silent‐site nucleotide diversity data (π_silent) can provide insights into the forces driving genome evolution. Here we present π_silent statistics for the mitochondrial and nuclear DNAs of Polytomella parva, a nonphotosynthetic green alga with a highly reduced, linear fragmented mitochondrial genome. We show that this species harbors very little genetic diversity, with the exception of the mitochondrial telomeres, which have an excess of polymorphic sites. These data are compared with previously published π_silent values from the mitochondrial and nuclear genomes of the model species Chlamydomonas reinhardtii and Volvox carteri, which are close relatives of P. parva, and are used to understand the modes and tempos of genome evolution within green algae.     

Epigenomic stability assessment during cryopreservation and physiology among various strains of Chromochloris zofingiensis (Chlorophyceae) and their genetic variability revealed by AFLP and MS-AFLP

Chromochloris zofingiensis (Dönz) Fucíková & L.A.Lewis, due to its production of highly valuable carotenoids such as astaxanthin, is a model organism in biotechnology. Since the recognition of this physiological property, many biotechnological applications have only used a single strain (SAG 211-14 = CCAP 211/14 = UTEX 32 = ATCC 30412) to produce biomass and carotenoids. However, multiple acquisitions of strains putatively belonging to the same species raised the question of the conspecificity of those strains and their properties. In this study, the conspecificity of the available strains, which are deposited axenically in SAG, was tested using SSU and ITS rDNA sequencing and AFLP (EcoRI/PstI) analyses. The comparison of SSU and ITS rDNA sequences as well as the AFLP patterns revealed that the investigated strains formed two very similar groups, (1) SAG 211-14, SAG 4.80, SAG 31.80, and SAG 34.80 and (2) SAG 221-2. All strains belonged to one species, C. zofingiensis, and represented one monophyletic lineage within the so-called DO-group of the Chlorophyceae. The robustness to cryopreservation and the subsequent epigenetic variability was detected using the methylation-sensitive AFLP (EcoRI/MspI and EcoRI/HpaII) among the five Chromochloris strains. All strains showed a high rate of survival (54.4–98.1%) during cryopreservation. The methylation patterns varied between precryo and postcryo in all strains detected among three time points (before, shortly after, and 8 weeks after cryopreservation), showing that the MS-AFLP technique has the potential to detect epigenetic effects occurring in response to cryopreservation and other stresses. Finally, the potential of these five strains for usage in biotechnological applications was proven by growing them in aerated cultures with and without additional carbon dioxide supply. The comparison showed that all strains produced high amounts of biomass and carotenoids under aeration with additional CO₂ and were therefore suitable in biotechnology.

     

A global molecular phylogeny of 147 periwinkle species (Gastropoda,Littorininae)

Reid, D. G., Dyal, P. & Williams, S.T. (2012) A global molecular phylogeny of 147 periwinkle species (Gastropoda, Littorininae). —Zoologica Scripta, 41, 125–136. Complete species‐level molecular phylogenies have been published for several genera of Littorinidae (e.g. Echinolittorina, Littoraria). Here we add new sequence data from three genes (28S rRNA, 12S rRNA, cytochrome oxidase c subunit I) for single specimens of an additional 24 species, to make a data set of 147 (97%) of the 152 recognized species of the subfamily Littorininae. This three‐gene data set is analysed to produce a phylogenetic hypothesis for the subfamily, which includes the first complete species‐level phylogeny of the genus Peasiella and the first three‐gene phylogeny of all Littorina species. The non‐planktotrophic species of Littorina have previously been classified together (as subgenus Neritrema), implying a single origin of this developmental mode. Tests of this hypothesis with the new data are inconclusive, and resolution is not improved in a tree constructed from five genes (adding previously published sequences of 16S rRNA and cytochrome b). Using available fossils for calibration we generate a BEAST chronogram, which emphasizes that the radiation of Littorina is more recent than that of other littorinine genera. A database is provided, listing all known species of Littorininae, with their distributions, development, ecology and gene sequences, as a tool for future evolutionary studies of this model group.     

Recombinant Protein Secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a Novel Signal Peptide

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.     

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.