Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.     

PHYLOGENETIC ANALYSIS OF THE GENUS EUGLENA (EUGLENOPHYCEAE) WITH PARTICULAR REFERENCE TO THE TYPE SPECIES EUGLENA VIRIDIS1

Euglena viridis (subgenus Euglena) serves as the type species for the genus Euglena. In this study, molecular phylogenetic analyses using a small subunit (SSU) and a combined SSU–partial large subunit rDNA data set for members of the genus Euglena showed that strains identified as E. viridis on the basis of morphology are distributed between two separate nonsister clades. Although all the E. viridis strains examined were morphologically indistinguishable and possessed spherical mucocysts and stellate chloroplasts with one paramylon center, there was a high degree of sequence divergence between the E. viridis strains in different clades, making this a cryptic species. Like E. viridis, all taxa from the subgenus Euglena are characterized by having one or more stellate chloroplasts with paramylon grains clustered around the center of the chloroplast. These additional taxa were divided into four clades in all the molecular analyses. Strains of Euglena stellata formed two nonsister clades whose members had a single aggregate chloroplast with paramylon center and spindle‐shaped mucocysts. A geniculata clade included species with one or two stellate chloroplasts with paramylon centers and spherical mucocysts, and the cantabrica clade had members with one stellate chloroplast with paramylon center and spherical mucocysts often arranged in spiral rows. Interspersed among these were three additional clades bearing taxa from the subgenus Calliglena that contains members with discoid plastids and pyrenoids that may or may not be capped with paramylon. These taxa formed a laciniata clade, mutabilis clade, and gracilis clade. This study demonstrates that E. viridis and E. stellata are cryptic species that can only be distinguished at the molecular level. Because E. viridis is the designated type species for the genus Euglena, we designated an epitype for E. viridis.     

Australian Laurencia majuscula (Rhodophyta,Rhodomelaceae) and the Brazilian Laurencia dendroidea are conspecific

Morphological and molecular studies have been undertaken on two species of the red algal genus Laurencia J.V.Lamouroux: Laurencia majuscula (Harvey) A.H.S. Lucas and Laurencia dendroidea J.Agardh, both from their type localities. The phylogenetic position of these species was inferred by analysis of the chloroplast‐encoded rbcL gene sequences from 24 taxa. In all phylogenetic analyses, the Australian Laurencia majuscula and the Brazilian L. dendroidea formed a well‐supported monophyletic clade within the Laurencia sensu stricto. This clade was divided into two subclades corresponding to each geographical region; however, the genetic divergence between Australian L. majuscula and Brazilian L. dendroidea was only 0–1.35%. Examination of the type specimens and sequences of freshly collected samples of both Laurencia majuscula and L. dendroidea show the two to be conspecific despite their disjunct type localities.     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

MOLECULAR PHYLOGENY OF SELECTED MEMBERS OF THE ORDER THALASSIOSIRALES (BACILLARIOPHYTA) AND EVOLUTION OF THE FULTOPORTULA1

Recent phylogenetic studies of the diatoms indicate that members of the order Thalassiosirales occupy an interesting position in the diatom evolutionary tree. Despite their radial morphology and scaly auxospores, they are consistently recovered in molecular analyses as a member of subdivision Bacillariophytina and a sister clade to non‐fultoportulate and non‐radial lithodesmioids. This study included 46 species from nine traditionally accepted extant genera, and analyzed 43 nuclear small subunit (SSU) rRNA sequences in parallel with a survey of the variation in fultoportula structure. Three possible scenarios leading to the evolution of the fultoportula are discussed in the context of molecular and morphological similarities between the examined Thalassiosirales and their SSU rRNA sister clade Lithodesmiales. We speculate that the fultoportula might be derived by a modification of either a cribrum in an areola (fultoportula within an areola), or structures similar to marginal ridges now seen in lithodesmioids around a cluster of poroids (fultoportula in a tube), or finally, that the central fultoportula may have an origin different from the marginal fultoportulae. Our data confirm that fultoportula‐bearing diatoms constitute a natural phylogenetic group. The families Thalassiosiraceae, Skeletonemaceae, and Stephanodiscaceae and the genus Thalassiosira Cleve were unexpectedly found to be paraphyletic. Further, Cyclotella Kutz. and Stephanodiscus Ehr. may not be closely related and some species of these genera are more closely allied to other species of Thalassiosira. The generitype, T. nordenskioeldii, is embedded within a large poorly structured cluster of species that includes several members of Thalassiosira, Planktoniella sol, Minidiscus trioculatus, and two members of Stephanodiscus. An emendment of the order Lithodesmiales and the family Lauderiaceae are proposed.     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

EVOLUTIONARY ASSOCIATIONS OF BROOD PARASITIC FINCHES (VIDUA) AND THEIR HOST SPECIES: ANALYSES OF MITOCHONDRIAL DNA RESTRICTION SITES

The species-specific associations of the African brood parasitic finches Vidua with their estrildid finch host species may have originated by cospeciation with the host species or by later colonizations of new hosts. Predictions of these alternative models were tested in two species groups of brood parasites (indigobirds, paradise whydahs) and their hosts. Phylogenetic analyses suggested that the brood parasites and their hosts did not speciate in parallel. The parasitic indigobirds share mitochondrial haplotypes with each other, and species limits in both indigobirds and paradise whydahs do not correspond with their gene trees. Different parasite species within a region are more closely related to each other than any is to parasites that are associated with its same host species in other regions of Africa. There is little genetic difference between parasite species D?_i,j < 0.001 in the indigobirds, D?_i,j = 0.01 in the whydahs). Genetic distances D?_i,j between the parasite species are less than the genetic distances between their corresponding host species in all parasite-host comparisons, and average only 7.2% as large in the indigobirds as in their hosts and 42% as large in the paradise whydahs as in their hosts. A phylogenetic model that allows ancestral haplotype polymorphisms to be retained in descendant species was compared to a constraint model of species monophyly requiring all but the one ancestral haplotype to be independently derived within each species. The constraint model increases the length of the indigobird tree by 50% over that of the model of retained ancestral polymorphisms; the difference is statistically significant. Both phylogenetic and distance analyses indicate that the brood parasites have become associated with their host species through host switches and independent colonizations of the hosts, rather than through parallel cospeciation with them. The molecular genetic results are supported by recent discoveries of additional host species that are associated with the indigobirds in the field and by variation in the species-specific song behaviors of the brood parasites.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

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.     

Morphology of Laurencia clavata and L. elata (Ceramiales,Rhodophyta) in relation to generic circumscription in the Laurencia complex

Laurencia Lamouroux (Rhodophyta) was recently separated into three genera–Laurencia, Chondrophycus (Tokida et Saito) Garbary et Harper and Osmundea Stackhouse – each of which was newly defined based on vegetative and reproductive structures. In this study, the previously unknown vegetative and reproductive morphology of two Australian endemic species of Laurencia, L. clavata Sonder and L. elata (C. Agardh) Harvey, was studied, particularly in the context of the revised generic delineation. These species exhibit vegetative axes with four pericentral cells and trichoblast-type spermatangial development. Tetrasporangia are abaxially produced from the existing third and fourth pericentral cells. L. clavata has terete thalli with distinctive verticillate branching and is similar to Chondria C. Agardh, rather than to Laurencia, in having an unusually marked constriction at the base of the branches and starch accumulation in subcortical and medullary cells. Compared to Laurencia, apical cells of this species exhibit a less oblique division ; the resulting recognizable axial cell rows extend somewhat below the branches, and particularly at a young stage they are also clear throughout branchlets. However, other vegetative and spermatangial structures show that L. clavata is more closely allied to Laurencia than to Chondria, and it is placed in Laurencia. By contrast, L. elata exhibits morphology typical of Laurencia and is characterized by large, robust, compressed thalli with fastigiately distichous branching and an extensive secondary cortex. Furthermore, it appears to be distinct from similar species in sometimes having a parasitic species of Janczewskia Solms-Laubach (Rhodophyta). Taxonomy of Laurencia is discussed on the basis of these and previous studies.     

Phylogeny,Evidence for a Cryptic Plastid,and Distribution of Chytriodinium Parasites (Dinophyceae) Infecting Copepods

Spores of the dinoflagellate Chytriodinium are known to infest copepod eggs causing their lethality. Despite the potential to control the population of such an ecologically important host, knowledge about Chytriodinium parasites is limited: we know little about phylogeny, parasitism, abundance, or geographical distribution. We carried out genome sequence surveys on four manually isolated sporocytes from the same sporangium, which seemed to be attached to a copepod nauplius, to analyze the phylogenetic position of Chytriodinium based on SSU and concatenated SSU/LSU rRNA gene sequences, and also characterize two genes related to the plastidial heme pathway, hemL and hemY. The results suggest the presence of a cryptic plastid in Chytriodinium and a photosynthetic ancestral state of the parasitic Chytriodinium/Dissodinium clade. Finally, by mapping Tara Oceans V9 SSU amplicon data to the recovered SSU rRNA gene sequences from the sporocytes, we show that globally, Chytriodinium parasites are most abundant within the pico/nano‐ and mesoplankton of the surface ocean and almost absent within microplankton, a distribution indicating that they generally exist either as free‐living spores or host‐associated sporangia.     

TAXONOMY OF THE PHACUS OSCILLANS (EUGLENACEAE) AND ITS CLOSE RELATIVES—BALANCING MORPHOLOGICAL AND MOLECULAR FEATURES1

The establishment of epitypes (together with emended diagnoses) for seven species of Phacus Dujard. [Phacus oscillans G. A. Klebs, Phacus parvulus G. A. Klebs, Phacus pusillus Lemmerm., Phacus skujae Skvortzov, Phacus inflexus (Kisselew) Pochm., Phacus polytrophos Pochm., and Phacus smulkowskianus (Zakry?) Kusber] was achieved by literature studies, verification of morphological diagnostic features (cell size, cell shape), as well as molecular characters (SSU rDNA). The investigated Phacus species are mostly well distinguished morphologically, with an SSU rDNA interspecific sequence similarity of 95.1%–99.0% and an intraspecific sequence similarity of 99.0%–99.9%. Some of the phylogenetic relationships among the seven species have not been resolved, but the topology obtained indicates their assignment into two sister clades. The first clade is composed of two sister groups (P. parvulus and P. pusillus), while the second constitutes an assemblage of the remaining five species. The relationships between the clades remain unresolved.     

THERMAL ECOPHYSIOLOGY OF LAURENCIA PACIFICA AND LAURENCIA NIDIFICA (CERAMIALES,RHODOPHYTA) FROM TROPICAL AND WARM‐TEMPERATE REGIONS1

Laurencia is a globally distributed genus with about 80 species (order Ceramiales) that inhabit tropical, subtropical, and warm‐temperate regions of both sides of the Atlantic and Indo‐Pacific oceans. This study investigated how two species of Laurencia distributed in different thermal environments (California and Hawaii) varied in their photosynthetic responses to temperature. The thermal ecophysiology of both species was investigated at different temporal scales (short‐term responses and seasonal acclimatization) using oxygen evolution and pulse‐amplitude‐modulated (PAM) fluorometry. Our results indicated that seasonal acclimatization of both species of Laurencia influenced the short‐term photosynthetic response at both locations. Greater seasonal differences in the photosynthetic performance were observed for L. pacifica Kylin, which reflects the ability of this species to acclimatize to local environmental conditions characterized by short‐term fluctuations and a broader annual temperature range. Photosynthetic performance of L. nidifica J. Agardh was consistent with the less variable local environment (no short‐term fluctuations and a narrower temperature range). These results suggest that acclimatization to temperature variability in the environment can influence the degree of flexibility of physiological responses of species in this genus.     

LAURENCIA MARILZAE SP. NOV. (CERAMIALES,RHODOPHYTA) FROM THE CANARY ISLANDS,SPAIN, BASED ON MORPHOLOGICAL AND MOLECULAR EVIDENCE1

Laurencia marilzae Gil‐Rodríguez, Sentíes et M.T. Fujii sp. nov. is described based on specimens that have been collected from the Canary Islands. This new species is characterized by distinctive yellow–orange as its natural habitat color, a terete thallus, four pericentral cells per vegetative axial segment, presence of secondary pit‐connections between adjacent cortical cells, markedly projecting cortical cells, and also by the presence of corps en cerise (one per cell) present in all cells of the thallus (cortical, medullary, including pericentral and axial cells, and trichoblasts). It also has a procarp‐bearing segment with five pericentral cells and tetrasporangia that are produced from the third and fourth pericentral cells, which are arranged in a parallel manner in relation to fertile branchlets. The phylogenetic position of this taxon was inferred based on chloroplast‐encoded rbcL gene sequence analyses. Within the Laurencia assemblage, L. marilzae formed a distinctive lineage sister to all other Laurencia species analyzed. Previously, a large number of unique diterpenes dactylomelane derivatives were isolated and identified from this taxon. L. marilzae is morphologically, genetically, and chemically distinct from all other related species of the Laurencia complex described.     

PHYLOGENY AND THE EVOLUTION OF HOST PLANT ASSOCIATIONS IN THE LEAF BEETLE GENUS OPHRAELLA (COLEOPTERA,CHRYSOMELIDAE)

Species of Ophraella, a North American genus of leaf beetles (Chrysomelidae), feed variously on eight genera in four tribes of Asteraceae. A phylogenetic analysis, based on morphological features and allozymes, was undertaken to deduce the history of host affiliation within the genus. The two data sets are combined to arrive at a provisional phylogeny of the species, onto which host associations are parsimoniously mapped. Among and within the 12 species studied, at least two shifts are postulated to have occurred among congeneric plant species, five between genera in the same tribe, and four between different tribes of Asteraceae. The phylogeny of Ophraella appears not to be congruent with that of its hosts. This and other evidence indicates that many host shifts in Ophraella postdate the divergence of the host plants, a conclusion that may apply commonly to phytophagous insects. A phenetic analysis of the plants' secondary compounds provides modest support for the hypothesis that host shifts are facilitated by commonalities in plant chemistry. A possible trend in host shifts is evident, from chemically simpler to chemically more forbidding plants. The chemical barriers to host shifts in Ophraella appear to require adaptation in both behavior and in physiological attributes. There is no evidence that the host associations of these insects or the divergence in secondary chemistry of their hosts can be attributed to coevolution.