Plastid psbA Research Articles

Sphaerocarpos ibericus sp. nov. (Sphaerocarpaceae), a new European liverwort species that includes S. stipitatus auct. eur. non Lindenb.

ABSTRACT Introduction Specimens of Sphaerocarpos collected during 2022 from the central Iberian Peninsula did not match any previously known species of this genus. Examination of other herbarium material revealed three identical specimens from three different localities in Spain. These were also morphologically identical to Portuguese specimens previously identified as S. stipitatus, a southern hemisphere and Himalayan species that in fact is different from these Portuguese plants. Methods The morphology of the newly collected specimens was compared with that of the known European species plus descriptions of South African plants of Sphaerocarpos stipitatus. A phylogenetic analysis based on the nuclear gene 26S and plastid psbA and rbcL regions was performed to investigate its relationships in the genus. Key results and conclusions Based on morphological and molecular data, we describe the plant as a new species of bryophyte, Sphaerocarpos ibericus M.Infante, Luceño, Quirós-de-la-Peña, Míguez, J.Muñoz & Heras, hitherto an Iberian endemic. It is diagnosed by having cylindrical female involucres with a partially bistratose wall whose inner cells are finger-like and arranged in more-or-less definite ribs in the medial part of the involucre, and the distal part of its spores covered with complete areolae larger at the centre of the spores than on their sides, gradually becoming incomplete and with the remaining lamellae transforming into ridges towards the line where the individual spores of the tetrad are joined. According to published illustrations, Portuguese specimens identified as S. stipitatus pertain indeed to the new species, and therefore S. stipitatus auct. eur. non Lindenb. should be removed from the European liverwort flora.

Unraveling Polyphyly in Flourensia (Asteraceae, Heliantheae, Enceliinae) and the Establishment of a New Genus Austroflourensia

The monophyly of Flourensia was examined for the first time by sequencing the nuclear ITS and plastid psbA- trnH regions in 18 species of the genus, analyzing them along with representatives of the remaining genera of subtribe Enceliinae. Results showed strong evidence for the polyphyly of Flourensia identifying two well-supported groups: Flourensia, a clade from North America including the type F. laurifolia, and another clade, here designed as the new genus Austroflourensia, containing the South American species. Austroflourensia is related to the other four genera composing the subtribe Enceliinae, whereas Flourensia s.s. is sister to all of them. Austroflourensia can be mainly distinguished by having a shrubby or subshrubby habit, capitula always radiate usually arranged in weakly cymose-corymbose capitulescences, phyllaries 2-3-seriate, and disc corollas shortly dentate. This paper proposes twelve new combinations to accommodate species previously described in the genus Flourensia and provides emended descriptions of Flourensia and the new genus Austroflourensia. The illustration of the type of the new genus, a distribution map, and a key to the genera of Enceliinae are also provided.

A REMARKABLE NEW GYNODIOECIOUS SPECIES OF OCOTEA (LAURACEAE) FROM THE BRAZILIAN ATLANTIC RAIN FOREST, AND ITS PHYLOGENETIC PLACEMENT IN THE OCOTEA COMPLEX

Ocotea bilocellata Baitello, D.B.O.S.Cardoso & P.L.R.Moraes, a species of Lauraceae that is new to science and from the Atlantic rain forest of the state of São Paulo, Brazil, is described and illustrated. The newly described species does not fit in any of the traditionally circumscribed disporangiate Lauraceae genera; rather, we show that it is phylogenetically placed within the Ocotea minarum group, as revealed by nuclear ribosomal ITS and plastid psbA–trnH sequence data.

Molecular diversity of the genus Cryptomonas (Cryptophyceae) in Russia

ABSTRACT Molecular data were used to revise the diversity of the genus Cryptomonas in Russia, using samples collected across nine geographic regions within Russia from 2015–2019. Several molecular markers were used: nuclear SSU, LSU and ITS2 rDNA, and plastid psbA. In total, 24 species of the genus Cryptomonas were identified in the country’s fresh waters based on 122 different strains. Six new species were identified and are described here (Cryptomonas matvienkoae sp. nov., C. furtiva sp. nov., C. paludosa sp. nov., C. ursina sp. nov., C. kisselevii sp. nov. and C. meshchorana sp. nov.), and three taxa are emended (Cryptomonas skujae, C. platyuris and C. obovata). These new and emended species group in clades with the previously described species or represent new lineages on the phylogenetic tree. Seven morphospecies, previously reported in Russia, are confirmed by molecular methods, and two unidentified taxa are also reported. HIGHLIGHTS Molecular investigations of the genus Cryptomonas in Russia revealed 24 species. Descriptions of three species were emended. Six species new to science were described.

Three new tribes in Myrtaceae and reassessment of Kanieae

The current tribal classification of Myrtaceae was based on analysis of the plastid matK coding region within the trnK intron. The phylogenetic position of the genera Cloezia and Xanthomyrtus was poorly supported, and the original sequence for Kania, the type genus of the tribe Kanieae, was rather poor. To clarify relationships, we sequenced plastid psbA–trnH and an extended portion of the trnK intron, including the spacer regions flanking matK, and nuclear ribosomal ITS and ETS regions for representative species across the tribes, including denser sampling of the three genera of interest. Analyses of these extended datasets show a strong relationship between Kania and the tribe Metrosidereae but not with other genera presently assigned to the Kanieae. The relationship between Kania and the tribe Metrosidereae is strongly correlated with morphological features recently documented in Metrosideros fossils. Consequently, a new tribe, Tristaniopsideae PeterG.Wilson, is described to accommodate most genera presently assigned to Kanieae. Furthermore, the morphological divergence and genetic distance shown by Cloezia and Xanthomyrtus are here considered as justifying their recognition as the tribes Cloezieae Peter G.Wilson and Xanthomyrteae Peter G.Wilson. Recognition of these tribes brings to four the number of tribes absent from present-day mainland Australia. Prior to this study, Metrosidereae was the only tribe in subfamily Myrtoideae that was absent from mainland Australia.

Cryptomonas indica sp. nov. (Cryptophyceae: Cryptomonadales), a new species described from the Western Ghats, India

A new species, Cryptomonas indica sp. nov., is described from Western Ghats, India, based on morphological and molecular data. Phylogenetic relationships inferred from nuclear small and large subunit ribosomal DNA, internal transcribed spacer 2, and plastid psbA sequences show that the new species forms a separate lineage on the phylogenetic tree of the genus Cryptomonas. This new species is included in the basal clade of Cryptomonas tree, which consists of undescribed taxa from Europe and South Korea. Cells of this species are up to 19 µm in length, elliptical to slightly asymmetrical in broad view, and have a plastid with four pyrenoids. Cryptomonas indica has been observed in one locality, the Northern region of the Western Ghats. Notably, this is the first Cryptomonas taxon described from the Indian subcontinent with both morphology and molecular information.

Monoclonal Culture and Characterization of Symbiodiniaceae C1 Strain From the Scleractinian Coral Galaxea fascicularis.

The symbiosis between cnidarian hosts and photosynthetic dinoflagellates of the family Symbiodiniaceae (i.e., zooxanthellae) provides the energy foundation of coral reef ecosystems in oligotrophic waters. The structure of symbiont biota and the dominant species of algal symbiont partly shape the environmental adaptability of coral symbiotes. In this study, the algal symbiont cells were isolated from the tentacles of Galaxea fascicularis, a hermatypic coral with obvious differentiation in heat resistance, and were cultured in vitro with an improved L1 medium. An algal monoclonal cell line was established using separated algal culture drops and soft agar plating method, and named by GF19C1 as it was identified as Cladocopium sp. C1 (Symbiodiniaceae) based on its ITS1, ITS2, and the non-coding region of the plastid psbA minicircle (psbAncr) sequences. Most GF19C1 cells were at the coccoid stage of the gymnodinioid, their markedly thickened (ca. two times) cell wall suggests that they developed into vegetative cysts and have sexual and asexual reproductive potential. The average diameter of GF19C1 cells decreased significantly, probably due to the increasing mitotic rate. The chloroplasts volume density of GF19C1 was significantly lower than that of their symbiotic congeners, while the surface area density of thylakoids relative to volumes of chloroplasts was not significantly changed. The volume fraction of vacuoles increased by nearly fivefold, but there was no significant change in mitochondria and accumulation bodies. Light-temperature orthogonal experiments showed that, GF19C1 growth preferred the temperature 25 ± 1°C (at which it is maintained post-isolation) rather than 28 ± 1°C under the light intensity of 42 ± 2 or 62 ± 2 μmol photons m–2 s–1, indicating an inertia for temperature adaptation. The optimum salinity for GF19C1 growth ranged between 28–32 ppt. The monoclonal culture techniques established in this study were critical to clarify the physiological and ecological characteristics of various algal symbiont species, and will be instrumental to further reveal the roles of algal symbionts in the adaptive differentiation of coral-zooxanthellae holobionts in future studies.

Corallina (Corallinales, Rhodophyta) from Tristan da Cunha and the Falkland Islands: implications for South Atlantic biogeography

ABSTRACT A molecular and morphological taxonomic study of Corallina (Corallinales, Rhodophyta) from Tristan da Cunha and the Falkland Islands revealed Corallina chamberlainiae J.Brodie & R.Mrowicki sp. nov. from both South Atlantic archipelagos, and Corallina cf. caespitosa only in the Falkland Islands. Analysis of mitochondrial COI-5P and plastid psbA resolved C. chamberlainiae as a distinct clade composed of specimens from Tristan and the Falkland Islands (COI-5P), in addition to two matching New Zealand samples (psbA). In psbA analyses, C. cf. caespitosa was close to two species from Japan (as C. pilulifera and C. melobesioides) but was separate from C. caespitosa sensu stricto from Britain and C. ferreyrae (isotype) from Peru. In COI-5P analyses, C. cf. caespitosa was in a clade with C. caespitosa (holotype) and close to, but distinct from, C. ferreyrae and an unidentified South African Corallina species. Application of the Automatic Barcode Gap Discovery (ABGD) and Poisson tree processes (bPTP) for COI-5P lumped C. chamberlainiae into one clade, whereas the Generalized Mixed Yule Coalescent (GMYC) model split it into two well-supported groups, one of which only contained Falkland Island specimens. All three models lumped C. cf. caespitosa with C. caespitosa. C. chamberlainiae closely resembles C. officinalis, the generitype, but is distinguished by smaller size and a more compressed thallus towards the apex. Corallina cf. caespitosa resembles C. caespitosa but is smaller and has fused, palm-like upper fronds. Neither of these taxa is conspecific with C. ferreyrae although C. caespitosa was recently synonymized with C. ferreyrae. C. chamberlainiae and C. cf. caespitosa occupy similar intertidal habitats to their respective, closely related counterparts C. officinalis and C. caespitosa. The results indicate cryptic diversity and suggest that there are many misidentified Corallina species. The paucity of South Atlantic studies of the Corallinales points to the need for a much greater taxonomic effort in this biogeographic region.

<p><strong><em>Cryptomonas cattiensis</em></strong><strong> <em>sp. nov.</em> (Cryptophyceae: Cryptomonadales), a new species described from Vietnam</strong></p>

A new species, Cryptomonas cattiensis sp. nov., is described from Vietnam based on morphological and molecular data. Phylogenetic relationships inferred from nuclear small and large subunit ribosomal DNA, internal transcribed spacer 2, and plastid psbA sequences show that the new species forms a separate branch on the phylogenetic tree of the genus Cryptomonas. This species has slightly sigmoid to elliptical cells in ventral view and a single plastid, deeply notched into several irregular lobes, with two pyrenoids. C. cattiensis has been observed in one locality in Cat Tien National Park.

Molecular phylogeny and biogeography of Ecklonia spp. (Laminariales, Phaeophyceae) in Japan revealed taxonomic revision of E. kurome and E. stolonifera

ABSTRACT Species level taxonomy and genetic structures of local populations of Ecklonia species (Laminariales, Phaeophyceae) distributed in northeastern Asia were examined using multigene molecular phylogeny of mitochondrial atp8–16S rDNA, cox1 and cox3, plastid atpB, psaA, psbA and rbcL gene sequences, and nuclear microsatellite markers. Japanese Ecklonia formed three to four clades with high to medium support (Groups 1, 2a, 2b, and 3). Group 1 was composed of morphospecies of E. radicosa with full statistical support. Groups 2a, 2b, and 3 were distinct but genetically close, and support for each clade was weak. Those genetic groups had distinct distributions, with some overlaps in contact regions in the Seto Inland Sea and vicinity. Microsatellite analyses suggested genetic exchange between Groups 2b and 3 in their contact region. In conclusion, E. radicosa was revealed to be an independent species, and synonymy between Eckloniopsis and Ecklonia was supported. In contrast, the independence of morphospecies E. cava, E. kurome, and E. stolonifera at species level was not supported. Instead, three genetic groups with separate distributional ranges were revealed. Therefore, we propose recognising E. cava, E. kurome, and E. stolonifera as a single species, and treating those three genetic groups as subspecies of E. cava, i.e. E. cava ssp. cava, E. cava ssp. kurome, and E. cava ssp. stolonifera. Furthermore, depending on the occurrence of stolons forming new sporophytes, i.e. rhizomatous holdfasts, two varieties, var. stolonifera and var. kuromeoides, are proposed in E. cava ssp. stolonifera.

A multigene molecular phylogeny of Eisenia reveals evidence for a new species, Eisenia nipponica (Laminariales), from Japan

ABSTRACTRecently, synonymizing Eisenia with Ecklonia was suggested based on a molecular phylogeny using the rbcL gene and ITS1–5.8S rDNA sequences. However, in a multigene molecular phylogeny based on mitochondrial cox1, cox3 genes and the atp8–16S rDNA region, as well as the plastid atpB, psaA, psbA and rbcL genes, Ecklonia spp. formed a monophyletic clade supported by high statistical values, and Eisenia spp. showed monophyly depending on analytical methods. The Japanese Eisenia species that used to be identified as Eisenia arborea (E. arborea sensu Arasaki) was shown to be genetically distant from E. arborea from the NE Pacific and E. bicyclis from Japan. Eisenia spp. were morphologically distinct in having a split meristematic zone in the mature thallus forming a dorsi-ventral blade with false branches. Therefore, we propose reinstatement of Eisenia as an independent genus and describe a new species, Eisenia nipponica (= E. arborea sensu Arasaki) from Japan. E. nipponica is distributed on the Pacific Coast of central Honshu, having a separate geographic range from E. bicyclis, which is distributed on the Pacific coast of eastern Honshu and the Sea of Japan coast of north-western Honshu and northern Kyushu.

Expression of uidA gene in tobacco transplastomic plants under the control of 5'-untranslated region of genomic RNA of potato virus Y

Regulation of the expression of chloroplast genes mainly occurs at the post-transcriptional level, in which 5'-untranslated regions (5'UTR) of plastid mRNA play a significant role. In transplastomic tobacco plants, we studied the effect of the 5’UTRs of the genomic RNA of potato Y-virus (5’PVY), as well as the 5’UTR of the plastid psbA gene (5’psbA) on the expression of the bacterial reporter uidA gene encoding β-glucuronidase (GUS). Genetic constructs containing the uidA gene were created under the control of identical transcriptional regulatory elements: the promoter and terminator of the psbA gene and two different 5’UTRs — 5’psbA and 5’PVY. Тobacco chloroplasts were transformed with these constructs by bioballistic method. Transplastomic lines showed approximately 24-fold differences in GUS activity. Replacing the natural 5’psbA with the heterologous 5’PVY resulted in a significant decrease in the synthesis of GUS protein. Key words: chloroplasts, protein synthesis, 5’UTR, transplastomic plants, reporter protein.

Holobiont Diversity in a Reef-Building Coral over Its Entire Depth Range in the Mesophotic Zone

Mesophotic reef-building coral communities (~30-120 m depth) remain largely unexplored, despite representing roughly three-quarters of the overall depth range at which tropical coral reef ecosystems occur. Although many coral species are restricted to shallow depths, several species occur across large depth ranges, including lower mesophotic depths. Yet, it remains unclear how such species can persist under extreme low-light conditions and how the different symbiotic partners associated with these corals contribute to facilitate such broad depth ranges. We assessed holobiont genetic diversity of the Caribbean coral Agaricia undata over depth in three localities of Colombia: San Andres Island (between 37 and 85 m), Cartagena (between 17 and 45 m) and Parque Nacional Natural Corales de Profundidad (between 77 and 87 m). We used a population genomics approach (NextRAD) for the coral host, and amplicon sequencing for the associated Symbiodinium (non-coding region of the plastid psbA minicircle) and prokaryotic (V4 region of the 16S rRNA gene) symbiont community. For the coral host, genetic structuring was only observed across geographic regions, but not between depths. Bayesian clustering and discriminant analysis of principal components revealed genetic structuring between the three regions, but not between shallow ( 60 m) depths. This pattern was confirmed when evaluating pairwise differentiation (FST) between populations, with much higher values between regions (0.0467-0.1034) compared to between depths [within location; -0.0075-(-0.0007)]. Symbiotic partners, including seven types of zooxanthellae and 325 prokaryotic OTUs, did not exhibit partitioning across depths. All samples hosted Symbiodinium clade C3 and the type C3psbA_e was present in all depths. Alpha microbial diversity was not significantly different between zones (upper vs. lower), which community composition between coral colonies was similar in the two zones (ANOSIM, R = -0.079, P > 0.05). The coral microbiome was dominated by Uncultured Betaproteobacteria in the order EC94 (16%), Unknown-Bacteria (15%), family Cenarchaeaceae (12 %), Burkholderiaceae (10%), and Hahellaceae (10%). The constant coral-holobiont composition along the studied depth range suggests that identity of the symbionts is not responsible for the coral's broad depth range and adaptation to low light environments.

Phylogenetic relationships and biogeography of Ptilophora (Gelidiales, Rhodophyta) with descriptions of P. aureolusa, P. malagasya, and P. spongiophila from Madagascar

The genus Ptilophora currently includes 16 species occurring mostly in subtidal habitats of the Indo-Pacific Ocean, but its global diversity and biogeography are poorly understood. We analyzed mitochondrial cox1, plastid rbcL and plastid psbA sequences from specimens collected in southern Madagascar during the 2010 Atimo Vatae expedition and studied their morphologies. Both morphological and molecular data sets demonstrated the presence of five species in southern Madagascar: Ptilophora hildebrandtii, P.pterocladioides, and three new species described here, P.aureolusa, P.malagasya, and P.spongiophila. Ptilophoraaureolusa is distinguished by its compound pinnae with uniformly spaced pinnules. Ptilophoramalagasya has an indistinct midrib and irregularly spaced pinnules. Ptilophoraspongiophila, heavily coated with sponges, has cylindrical to flattened main axes, lateral and surface proliferations, and spatulate tetrasporangial sori. The species of Ptilophora found in Madagascar are endemic, except P.hildebrandtii, which also occurs in eastern Africa. Ptilophora comprises four phylogenetic groups that map to eastern Australia, Japan, western Australia/Southeast Asia/Madagascar/eastern Africa, and Madagascar/eastern Africa/Aegean Sea. Biogeographical analysis revealed that the ancestor of Ptilophora originated in Australia, but most of the species radiated from Madagascar.

Phylogeny of the Neotropical sages (Salvia subg. Calosphace; Lamiaceae) and insights into pollinator and area shifts

Salvia subg. Calosphace (Lamiaceae, Lamiales) is a highly diverse clade endemic to the New World. The phylogenetic relationships of Calosphace have been previously investigated using DNA sequences of nuclear ITS region and plastid psbA–trnH intergenic spacer, but the resulting trees lack resolution and support for many clades. The present paper reassesses the phylogenetic relationships of subgenus Calosphace, including a broader taxon sampling, with a special focus on representing previously unsampled sections, and using an additional plastid marker (trnL–trnF region). Our results show increased resolution and overall patterns of support, recovering ten main clades. Within core Calosphace, the most inclusive of these main clades, 17 new subclades were identified. Of the 42 sections for which more than one species was analysed, only 12 are monophyletic. Our biogeographical analysis identified at least twelve migrations to South America from Mexican and Central American lineages, in agreement with previous suggestions of multiple origins of South American Calosphace diversity. This analysis also confirmed a colonization of the Antilles by Andean lineages. The reconstruction of ancestral states of pollination syndromes showed multiple shifts to ornithophily from melittophily and one reversal to the latter.

Bricklebush (Brickellia) phylogeny reveals dimensions of the great Asteraceae radiation in Mexico

Data from molecular phylogenetics were used to assess aspects of diversity and relationships in Brickellia, a large and widespread genus of Eupatorieae. The dataset included sequence data from nuclear ribosomal ITS, ETS, and plastid psbA–trnH regions. An initial question was to assess the monophyly of the genus and whether Barroetea, Phanerostylis, and Kuhnia should be recognized as separate from or included in Brickellia. The results supported the hypothesis that Brickellia is monophyletic, with the small (2–3 species) Pleurocoronis as the sister group and showed Barroetea, Phanerostylis, and Kuhnia all embedded within the genus. Results of a time calibrated phylogeny from a BEAST analysis gave an estimated origination time for Brickellia at about 9 million years ago (Ma), with the oldest split within the genus dated at about 7.5Ma. A BAMM analysis based on the time calibrated tree showed that Brickellia has one rate shift in diversification associated with its origin in the late Miocene. Some lineages within the genus have had an increase in the rate of diversification over the past 5Ma, whereas other lineages have had a decrease in net diversification during this period. The results also elucidated nine clades within Brickellia which are accepted as taxonomic sections, and that will form logical units for future detailed studies.

PsbA based molecular analysis of cross-feeding experiments suggests that Dinophysis acuta does not harbour permanent plastids

Toxic marine dinoflagellate species of the genus Dinophysis Ehrenberg are obligate mixotrophs that require feeding on the ciliate Mesodinium rubrum and light to achieve growth. It is now well known that they harbour plastids of cryptophyte origin, particularly of the genus Teleaulax, Plagioselmis or Geminigera group (TPG clade). Nevertheless, whether these plastids are permanent, or periodically acquired from M. rubrum prey, need additional studies in different phototrophic Dinophysis species. The origin of plastids from Dinophysis acuta Ehrenberg, one of the main agents of diarrhetic shellfish poisoning (DSP) outbreaks in Western Europe, was investigated here. Cross feeding-starvation experiments were carried out with cultures of D. acuta using M. rubrum as prey, the latter fed with two cryptophyte species, Teleaulax amphioxeia Hill and Teleaulax gracilis, belonging to the TPG clade in addition to Falcomonas sp. and Hemiselmis sp. The fate of cryptophyte plastids transferred to D. acuta through its ciliate prey was investigated using the plastid psbA gene as a tracer.

Ecological and biogeographic implications of Siderastrea symbiotic relationship with Symbiodinium sp. C46 in Sal Island (Cape Verde, East Atlantic Ocean)

The relative abundance of the genus Siderastrea and its relationship with temperature and irradiance was assessed around Sal Island (Cape Verde). In some of the surveyed sites, these corals accounted for 80–90 % of the living cover, making it a biotope-dominant organism. Unlike Siderastrea corals from West Atlantic and Caribbean locations, genetic analyses of the dinoflagellate symbiotic partner revealed high specificity between Siderastrea sp. in Cape Verde and the Symbiodinium type C46. Biotope restriction of the ecological success of Siderastrea in Cape Verde may be explained in part by this host–symbiont partnership, resulting locally in a small optimum ecological niche with specific light intensity regimes. Distinctively, West Atlantic and Caribbean Siderastrea associates with a much broader range of Symbiodinium diversity, suggesting that these symbioses exhibit some flexibility under differing environmental conditions where these corals occupy a wider range of ecological niches. Geographic isolation and/or long-standing environmental conditions are probably responsible for such adaptions and coral–dinoflagellate symbioses. Additional genetic analyses on Clade C Symbiodinium associated with Siderastrea were conducted with the hyper-variable plastid psbA minicircle to resolve phylogeographic patterns that indicate the relative connectivity and/or isolation of these symbionts throughout the tropical Atlantic.

Morphology and plastid psbA phylogeny of Zygnema (Zygnemataceae, Chlorophyta) from Korea: Z. insigne and Z. leiospermum

Zygnema is a conjugating filamentous green algal genus that is distributed in a broad range of freshwater habitats, from sea level to alpine summits. Although more than 150 species have been described worldwide, their taxonomy remains unclear, probably owing to their relatively simple morphology. We investigated the detailed morphology of Korean Zygnema species, combined with analysis of the plastid psbA gene from 22 specimens of the genus and putative relatives, in order to develope a key to their identification and isolation, and to determine their relationships. We recognized two species of Zygnema; Z. insigne and Z. leiospermum, based on morphological characters such as width of the vegetative cell, position of zygospores, dimensions and form of spores, shape of female gametangia, and color of mesospores. The analysis of psbA data was consistent with morphological comparison. The pairwise divergence between two species was 3.7-4.1% (34-38 bp) in psbA sequences. The phylogeny of psbA revealed the monophyly of Z. insigne and Z. leiospermum together with two isolates of Z. circumcarinatum from Germany and Scotland. This is the first report on the psbA gene phylogeny of Zygnema.

Improved Resolution of Reef-Coral Endosymbiont (Symbiodinium) Species Diversity, Ecology, and Evolution through psbA Non-Coding Region Genotyping

Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbAncr) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbAncr haplotype was recovered in most samples through direct sequencing (∼80–90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbAncr sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbAncr from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbAncr sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbAncr data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates.