Latitudinal and intracolony ITS-rDNA sequence variation in the symbiotic dinoflagellate genus Symbiodinium (Dinophyceae) in Zoanthus sansibaricus (Anthozoa: Hexacorallia)

We sequenced the internal transcribed spacer of ribosomal DNA (ITS‐rDNA) of Symbiodinium spp. (Freudenthal) from conspecific Zoanthus sansibaricus (Carlgren) colonies along a latitudinal gradient in Japan. Phylogenetic analysis reveals that Zoanthus in the two northern sites of Kokubu and Sakurajima harbor exclusively Symbiodinium subclade C1, whereas Yakushima Zoanthus harbors Symbiodinium subclades C1 and C15, and southernmost Amami Zoanthus Symbiodinium subclades A1 and C1, indicating holobiont flexibility. Individual Zoanthus colonies associated exclusively with one single subclade, but unexpectedly there was small variation between Symbiodinium ITS‐rDNA clone sequences obtained from within individual Zoanthus colonies. There was also a large deletion in the ITS‐2/28S rDNA boundary region in one clone sequence, and another large deletion in the 5.8S rDNA region in another clone. Our intracolony sequence heterogeneity might be a result of the presence of multiple copies of the ITS‐rDNA region within individual Symbiodinium genomes, or result from the possible presence of closely related Symbiodinium genotypes in the host.     

MITOCHONDRIAL DNA PHYLOGENY OF THE SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM,DINOPHYTA)1

Symbiotic dinoflagellates belonging to the genus Symbiodinium (Freudenthal) are found worldwide in association with shallow‐water tropical and subtropical marine invertebrates. Most phylogenetic studies of Symbiodinium have used nuclear rRNA (nrDNA) genes to infer relationships among members of the genus. In this report, we present the first phylogeny of Symbiodinium based on DNA sequences from a mitochondrial protein‐coding gene (cytochrome oxidase subunit I [cox1]). Two principal groups, one comprised of Symbiodinium clade A and the second encompassing Symbiodinium clades B/C/D/E/F, are strongly supported in the cox1 phylogeny. Relationships within Symbiodinium clades B/C/D/E/F, however, are less well resolved compared with phylogenies inferred from nrDNA and chloroplast large subunit (cp23S)‐rDNA genes. Statistical tests between alternative tree topologies verified, with an exception being the position of one controversial member of Symbiodinium clade D, that relationships inferred from cox1 are congruent with those inferred from nrDNA and cp23S‐rDNA. Taken together, the relationships between the major Symbiodinium clades are robust, and there appears to be no evidence of hybridization or differential introgression of nuclear and plastid genomes between clades.     

Molecular identification of symbiotic dinoflagellates (Symbiodinium spp.) from Palythoa spp. (Anthozoa: Hexacorallia) in Japan

In Japan, zooxanthellate Palythoa tuberculosa Klunzinger and Palythoa mutuki Verrill (Anthozoa: Hexacorallia: Zoantharia) are found over a 1,000 + km latitudinal range, often in environments where most other zooxanthellate anthozoans are not found (i.e. tidal lagoon pools, around shallow water hydrothermal vents, subtropical rocky shorelines). Sequences of internal transcribed spacer of ribosomal DNA (ITS-rDNA) of the symbiotic dinoflagellate genus Symbiodinium (zooxanthellae) Freudenthal (Order Suessiales) from P. tuberculosa and P. mutuki from several locations in Japan (34°11′N–24°16′N) were analysed. Unexpectedly, despite the ability of the genus Palythoa to be flexible in association with different Symbiodinium subclades, most (35 of 36) Palythoa investigated here specifically associate with subclade C1 and closely related types. Symbiodinium subclade C1 has been characterized as a “generalist” in terms of the ability to associate with a range of hosts, but present results suggest that subclade C1 may also be a “generalist” in terms of being able to live in a variety of environments over a latitudinal range. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Antigenic variation in mucilage secreted by members of the genus Symbiodinium (Dinophyceae)

Symbiodinium reside intracellularly in a complex symbiosome (host and symbiont‐derived) within cnidarian hosts in a specific host‐symbiont association. Symbiodinium is a diverse genus with variation greater than other dinoflagellate orders. In this paper, our investigation into specificity examines antigenic variation in the algal mucilage secretions at the host‐symbiont interface. Cultured Symbiodinium from a variety of clades were labeled with one of two antibodies to symbiont mucilage (PC3, developed using a clade B alga cultured from Aiptasia pallida; BF10, developed using a clade F alga cultured from Briareum sp.). The labeling was visualized with a fluorescent marker and examined with epifluorescence and confocal microscopy. PC3 antigen was found in cultured Symbiodinium from clades A and B, but not clades C, D, E and F. The correlation between labeling and clade may account for some of the specificity between host and symbiont in the field. Within clades A and B there was variation in the amount of label present. BF10 antigen was more specific and only found in cultures of the same cp23S‐rDNA strain the antibody was created against. These results indicate that the mucilage secretions do vary both qualitatively and quantitatively amongst Symbiodinium strains. Since the mucilage forms the host‐symbiont interface, variation in its molecular composition is likely to be the source of any signals involved in recognition and specificity.     

On the evolution and diversification of an Andean clade of reptiles: combining morphology and DNA sequences of the palluma group (Liolaemidae: Phymaturus)

Phymaturus comprises 44 species mainly distributed along the south‐west of South America on both sides of the Andes. In this study we present a phylogenetic analysis of Phymaturus of the palluma group, one of its two large clades, including almost all described species. This analysis duplicates the number of in‐group taxa compared with previous contributions. We performed a total‐evidence analysis, combining molecular and morphological characters: sequencing fragments of cytochome b (cytb), 12S, and ND4, for all terminals; describing 45 new morphological characters; and incorporating all DNA sequences available from GenBank. Separate analyses of morphology and DNA partitions are presented and discussed in detail. Seven subclades are recognized here. We named three new subclades and redefined another, found to be paraphyletic. In order to recognize lineages within the traditional Phymaturus palluma group we proposed to treat it as a natural group, containing within it the ranks of clade, subclade, and lineages, respectively. The palluma group is composed by the vociferator and the bibronii clades. The vociferator clade, composed of Chilean and Argentinean species, would be the most basal in the group. Within the bibronii clade, the roigorum subclade includes the Phymaturus verdugo lineage, whereas the mallimaccii subclade would consist of 13 terminal taxa, for which three Chilean species have been added. In this study, morphological apomorphies are identified for all clades and the evolution of ‘male head melanism’ is discussed. © 2015 The Linnean Society of London     

Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing

Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000‐fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral‐algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine‐scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral‐algal symbiosis.     

VARIABILITY IN THE PRIMARY SITE OF PHOTOSYNTHETIC DAMAGE IN SYMBIODINIUM SP. (DINOPHYCEAE) EXPOSED TO THERMAL STRESS1

Exposure to elevated temperature is known to cause photosynthetic inhibition in the coral symbiont Symbiodinium sp. Through the use of the artificial electron acceptor, methyl viologen, this study identified how reduced photosynthetic capacity occurs as a result of inhibition up‐ and/or downstream of ferredoxin in Symbiodinium sp. in hospite and in culture. Heterogeneity between coral species and symbiont clades was identified in the thermal sensitivity of photosynthesis in the symbionts of the scleractinian corals Stylophora pistillata and Pocillopora damicornis, as well as among Symbiodinium cultures of clades A, B, and C. The in hospite symbionts of S. pistillata and the cultured clade C Symbiodinium both exhibited similar patterns in that their primary site of thermal inhibition occurred downstream of ferredoxin at 32°C. In contrast, the primary site of thermal inhibition occurred upstream of ferredoxin in clades A and B at 32°C, while at 34°C, all samples showed combined up‐ and downstream inhibition. Although clade C is common to both P. damicornis and S. pistillata, the manner of thermal inhibition was not consistent when observed in hospite. Results showed that there is heterogeneity in the primal site of thermal damage in Symbiodinium among coral species and symbiont clades.     

Biosystematic studies on the genus Heloniopsis (Melanthiaceae) I. Phylogeny inferred from plastid DNA sequences and taxonomic implications

Based on a combined dataset of plastid DNA sequences (atpB‐rbcL, trnG, trnL‐trnL‐trnF, trnK 5' intron and matK) from 60 individuals, we conducted parsimony and likelihood analyses to clarify the phylogenetic relationships among the six species and three varieties that are commonly recognised in Heloniopsis, in addition to the related genera Ypsilandra and Helonias, using Chamaelirium and Chionographis as an outgroup. According to the single most parsimonious tree, which was identical to the maximum‐likelihood tree in topology, Helonias, Ypsilandra and Heloniopsis are all monophyletic with 100% bootstrap support (BS). In Heloniopsis, there are two highly supported clades (BS 94–97%): a clade of Korean species and a clade of Japanese and Taiwanese species. The latter clade comprised the following four subclades (BS 99–100%): 1) H. orientalis var. orientalis, 2) H. orientalis var. breviscapa and var. flavida, 3) H. kawanoi and 4) H. leucantha and H. umbellata. Because subclades 1 and 2 did not form a monophyletic group, and do show clear morphological differences – including nectary position, nectary‐sac structure and leaf margin undulation – they should be distinguished at the species level: H. orientalis for subclade 1 and H. breviscapa for subclade 2. In subclade 2, neither var. breviscapa nor var. flavida was monophyletic; instead, var. breviscapa plus var. flavida (thick‐leaved entity) was monophyletic (BS 62–63%) and var. flavida (thin‐leaved entity) was monophyletic (BS 86–87%). As var. breviscapa and var. flavida (thick‐leaved entity) share basally ± pinkish wide tepals and dark‐coloured thick leaves, in contrast to var. flavida (thin‐leaved entity), which has completely white narrow tepals and light‐coloured thin leaves, the two varieties should may be kept distinct after the merge of var. flavida (thick‐leaved entity) with var. breviscapa.     

SYMBIODINIUM NATANS SP. NOV.: A “FREE‐LIVING” DINOFLAGELLATE FROM TENERIFE (NORTHEAST‐ATLANTIC OCEAN)1

We examined a free‐living Symbiodinium species by light and electron microscopy and nuclear‐encoded partial LSU rDNA sequence data. The strain was isolated from a net plankton sample collected in near‐shore waters at Tenerife, the Canary Islands. Comparing the thecal plate tabulation of the free‐living Symbiodinium to that of S. microadriaticum Freud., it became clear that a few but significant differences could be noted. The isolate possessed two rather than three antapical plates, six rather than seven to eight postcingular plates, and finally four rather than five apical plates. The electron microscopic study also revealed the presence of an eyespot with brick‐shaped contents in the sulcal region and a narrow anterior plate with small knob‐like structures. Bayesian analysis revealed the free‐living Symbiodinium to be a member of the earliest diverging clade A. However, it did not group within subclade A_I (=temperate A) or any other subclades within clade A. Rather, it occupied an isolated position, and this was also supported by sequence divergence estimates. On the basis of comparative analysis of the thecal plate tabulation and the inferred phylogeny, we propose that the Symbiodinium isolate from Tenerife is a new species (viz. S. natans). To elucidate further the species diversity of Symbiodinium, particularly those inhabiting coral reefs, we suggest combining morphological features of the thecal plate pattern with gene sequence data. Indeed, future examination of motile stages originating from symbiont isolates will demonstrate if this proves a feasible way to identify and characterize additional species of Symbiodinium and thus match ribotypes or clusters of ribotypes to species.     

CAROTENOID COMPOSITIONS OF CLADOPHORA BALLS (AEGAGROPILA LINNAEI) AND SOME MEMBERS OF THE CLADOPHORALES (ULVOPHYCEAE,CHLOROPHYTA): THEIR TAXONOMIC AND EVOLUTIONARY IMPLICATION1

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the α‐carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila‐clade (clade 2), and divided clade 1 into subclade 1‐1 and subclade 1‐2. All the examined species positioned in the Aegagropila‐clade and those of the subclade 1‐1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1‐2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales. Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1‐2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.     

Surviving glacial ages within the Biotic Gap: phylogeography of the New Zealand cicada Maoricicada campbelli

Aim New Zealand is an ideal location in which to investigate the roles of landscape and climate change on speciation and biogeography. An earlier study of the widespread endemic cicada Maoricicada campbelli (Myers) found two phylogeographically distinguishable major clades – northern South Island plus North Island (northern‐SI + NI) and Otago. These two clades appeared to have diverged on either side of an area of the South Island known as the Biotic Gap. We sampled more intensively to test competing theories for this divergence. We aimed to discover if M. campbelli had survived within the Biotic Gap during recent glacial maxima, and if predicted areas of secondary contact between the two major clades existed. Location New Zealand. Methods We analysed mitochondrial DNA sequences (1520 bp; 212 individuals; 91 populations) using phylogenetic (maximum likelihood, Bayesian), population genetic (analysis of molecular variance) and molecular dating methods (Bayesian relaxed clock with improved priors). Results We found strong geographical structuring of genetic variation. Our dating analyses suggest that M. campbelli originated 1.83–2.58 Ma, and split into the two major clades 1.45–2.09 Ma. The main subclades in the northern‐SI + NI clade arose almost simultaneously at 0.69–1.03 Ma. Most subclades are supported by long internal branches and began to diversify 0.40–0.78 Ma. We found four narrow areas of secondary contact between the two major clades. We also found a difference between calling songs of the Otago vs. northern‐SI + NI clades. Main conclusions Phylogeographical patterns within M. campbelli indicate an early Pleistocene split into two major clades, followed by late Pleistocene range expansion and in situ population differentiation of subclades. The northern‐SI + NI clade diversified so rapidly that the main subclade relationships cannot be resolved, and we now have little evidence for a disjunction across the Biotic Gap. Structure within the main subclades indicates rapid divergence after a common bottlenecking event, perhaps attributable to an extremely cold glacial maximum at c. 0.43 Ma. Clade structure and dating analyses indicate that M. campbelli survived in many refugia during recent glacial maxima, including within the Biotic Gap. The narrow overlap between the two major clades is attributed to recent contact during the current interglacial and slow gene diffusion. The two major clades appear to be in the early stages of speciation based on genetic and behavioural differences.     

Acquisition of symbiotic dinoflagellates (Symbiodinium) by juveniles of the coral Acropora longicyathus

Scleractinian corals may acquire Symbiodinium from their parents (vertically) or from the environment (horizontally). In the present study, adult colonies of the coral Acropora longicyathus from One Tree Island (OTI) on the southern Great Barrier Reef (Australia) acquired two distinct varieties of symbiotic dinoflagellates (Symbiodinium) from the environment. Adult colonies had either Symbiodinium from clade C (86.7%) or clade A (5.3%), or a mixture of both clades A and C (8.0% of all colonies). In contrast, all 10-day-old juveniles were associated with Symbiodinium from clade A, while 83-day-old colonies contained clades A, C and D even though they were growing at the same location. Symbiodinium from clade A were dominant in both 10- and 83-day-old juveniles (99 and 97% of all recruits, respectively), while clade D was also found in 31% of 83-day-old juveniles. Experimental manipulation also revealed that parental association (with clade A or C), or the location within the OTI reef, did not influence which clade of symbiont was acquired by juvenile corals. The differences between the genetic identity of populations of Symbiodinium resident in juveniles and adult A. longicyathus suggest that ontogenetic changes in the symbiosis may occur during the development of scleractinian corals. Whether or not these changes are due to host selective processes or differences in the physical environment associated with juvenile versus adult colonies remains to be determined.     

Many corals host thermally resistant symbionts in high-temperature habitat

Physiologically distinct lines of dinoflagellate symbionts, Symbiodinium spp., may confer distinct thermal tolerance thresholds on their host corals. Therefore, if a coral can alternately host distinct symbionts, changes in their Symbiodinium communities might allow corals to better tolerate increasing environmental temperatures. However, researchers are currently debating how commonly coral species can host different symbiont types. We sequenced chloroplast 23 s rDNA from the Symbiodinium communities of nine reef-building coral species across two thermally distinct lagoon pools separated by ~500 m. The hotter of these pools reaches 35°C in the summer months, while the other pool’s maximum temperature is 1.5°C cooler. Across 217 samples from nine species, we found a single haplotype in both Symbiodinium clades A and D, but four haplotypes in Symbiodinium clade C. Eight of nine species hosted a putatively thermally resistant member of clade D Symbiodinium at least once, one of which hosted this clade D symbiont exclusively. Of the remaining seven that hosted multiple Symbiodinium types, six species showed higher proportions of the clade D symbiont in the hotter pool. Average percentage rise in the frequency of the clade D symbiont from the hotter to cooler pool was 52% across these six species. Even though corals hosted members of both the genetically divergent clades D and C Symbiodinium, some showed patterns of host–symbiont specificity within clade C. Both Acropora species that hosted clade C exclusively hosted a member of sub-clade C2, while all three Pocillopora species hosted a member of sub-clade C1 (sensu van Oppen et al. 2001). Our results suggest that coral–algal symbioses often conform to particular temperature environments through changes in the identity of the algal symbiont.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Symbiodinium diversity in the soft coral Heteroxenia sp. and its nudibranch predator Phyllodesmium lizardensis

We examined the diversity of the photosynthetic dinoflagellate, Symbiodinium, over a 2-year period in two invertebrates from Australia’s Northern Great Barrier Reef: the nudibranch Phyllodesmium lizardensis and an octocoral of the genus Heteroxenia. In years one and two, we used denaturing gradient gel electrophoresis with internal transcribed spacer 2 (ITS2) region amplicons and identified two nearly identical genotypes of clade C (C64 and a variant) in all samples of each species. We examined the secondary structure of both sequences and found that each had predicted ∆G values within the range of stable free energy values for Symbiodinium ITS2 sequences. In year two, we also used real-time quantitative polymerase chain reaction assays (qPCR) with clade-specific internal transcribed spacer 1 primers to determine whether there were cryptic clades (A, B, and/or D) associated with either host in addition to clade C. qPCR revealed that clades B, C, and D were present in all animals of both species and that all but two nudibranch samples also harbored clade A. These findings suggest that there may be more flexibility in this host/symbiont interaction than has previously been assumed.

     

Molecular evolution of a family of resistance gene analogs of nucleotide-binding site sequences in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Nucleotide-binding site-leucine-rich repeats (NBS-LRR) gene families are one of the major plant resistance genes. Genomic NBS evolution was studied in many plant species for diverse arrays of NBS gene families. In this study, we focused on one family of NBS sequences in an attempt to understand how closely related NBS sequences evolved in the light of selection in domesticated plant species. A phylogenetic analysis revealed five major clades (A–E) and five subclades (A1–A5) within clade A of cloned NBS sequences. Positive selection was only detected in newly evolved NBS lineages in subclades of clade A. Positively selected codon sites were found among NBS sequences of clade A. A sliding-window analysis revealed that regions with Ka/Ks ratios of >1 were in the inter-motifs when paired clades were compared, but regions with Ka/Ks ratios of >1 were found across NBS sequences when subclades of clade A were compared. Our results based on a family of closely related NBS sequences showed that positive selection was first exerted on specific lineages across all NBS sequences after selective constraints. Subsequently, sequences with mutations in commonly conserved motifs were scrutinized by purifying selection. In the long term, conserved high frequency alleles in commonly conserved motifs and changes in inter-motifs were maintained in the investigated family of NBS sequences. Moreover, codons identified to be under positive selection in the inter-motifs were mainly located in regions involved in functions of ATP binding or hydrolysis.     

Separate Introns Gained within Short and Long Soluble Peridinin-Chlorophyll a-Protein Genes during Radiation of Symbiodinium (Dinophyceae) Clade A and B Lineages

Here we document introns in two Symbiodinium clades that were most likely gained following divergence of this genus from other peridinin-containing dinoflagellate lineages. Soluble peridinin-chlorophyll a-proteins (sPCP) occur in short and long forms in different species. Duplication and fusion of short sPCP genes produced long sPCP genes. All short and long sPCP genes characterized to date, including those from free living species and Symbiodinium sp. 203 (clade C/type C2) are intronless. However, we observed that long sPCP genes from two Caribbean Symbiodinium clade B isolates each contained two introns. To test the hypothesis that introns were gained during radiation of clade B, we compared sPCP genomic and cDNA sequences from 13 additional distinct Caribbean and Pacific Symbiodinium clade A, B, and F isolates. Long sPCP genes from all clade B/B1 and B/B19 descendants contain orthologs of both introns. Short sPCP genes from S. pilosum (A/A2) and S. muscatinei (B/B4) plus long sPCP genes from S. microadriaticum (A/A1) and S. kawagutii (F/F1) are intronless. Short sPCP genes of S. microadriaticum have a third unique intron. Symbiodinium clade B long sPCP sequences are useful for assessing divergence among B1 and B19 descendants. Phylogenetic analyses of coding sequences from four dinoflagellate orders indicate that introns were gained independently during radiation of Symbiodinium clades A and B. Long sPCP introns were present in the most recent common ancestor of Symbiodinium clade B core types B1 and B19, which apparently diverged sometime during the Miocene. The clade A short sPCP intron was either gained by S. microadriaticum or possibly by the ancestor of Symbiodinium types A/A1, A3, A4 and A5. The timing of short sPCP intron gain in Symbiodinium clade A is less certain. But, all sPCP introns were gained after fusion of ancestral short sPCP genes, which we confirm as occurring once in dinoflagellate evolution.     

Molecular phylogenetic analysis of Podostemaceae: implications for taxonomy of major groups

The river‐weed family Podostemaceae (c. 300 species in c. 54 genera) shows a number of morphological innovations to be adapted to its unusual aquatic habitat, and its unique or rare bauplan features have been reflected in the traditional (i.e. non‐molecular) classification recognizing numerous monotypic or oligospecific genera. The infrasubfamilial relationships of many genera remained unclear. The present study used molecular phylogenetic analysis of matK sequences for 657 samples (c. 132 species/c. 43 genera). The family was traditionally divided into three subfamilies (Podostemoideae, Tristichoideae and Weddellinoideae). American Podostemoideae were shown to be polyphyletic and divided into four clades, i.e. Ceratolacis, Diamantina, Podostemum and all other genera. Among the podostemoid clades, Diamantina was the first branching clade and a clade comprising Mourera and the Apinagia subclade was then sister to the remainder of the New World and Old World Podostemoideae with low statistic supports. The Old World Podostemoideae comprised four monophyletic clades, i.e. two African clades, one Madagascan clade and one Asian clade, although the relationships among these clades and American Ceratolacis and Podostemum were poorly resolved. African Podostemoideae were polyphyletic, with Saxicolella pro parte being weakly supported as sister to the remaining Old World Podostemoideae plus Ceratolacis and Podostemum. In contrast to the American and African clades, monophyly of four Asian subclades was well supported. Plants of Tristicha (Tristichoideae) and of Weddellina (Weddellinoideae), which are currently treated as monospecific, had great matK differentiation equivalent to at least interspecific variation. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 461–492.     

Affinities in C3Cyperus lineages (Cyperaceae) revealed using molecular phylogenetic data and carbon isotope analysis

Maximum likelihood and Bayesian analyses of nrDNA (ETS1f) and plastid DNA (rpl32‐trnL, trnH‐psbA) sequence data are presented for ‘C₃Cyperus’ (Cyperaceae). The term ‘C₃Cyperus’ indicates all species of Cyperus s.l. that use C₃ photosynthesis linked with eucyperoid vegetative anatomy. Sampling comprises 77 specimens of 61 different taxa, representing nearly all previously recognized subdivisions of C₃Cyperus and the segregate genera Courtoisina, Kyllingiella and Oxycaryum. According to our results, the Cyperus clade is divided in six well‐supported clades. The first of these clades (clade 1) forms three subclades largely corresponding to Cyperus sections Haspani, Incurvi and Diffusi. Clade 2 comprises the entirely New World C. section Luzuloidei sensu Denton (1978). Clade 3 is a highly diverse clade including two subclades: clade 3a, C. sections Pseudanosporum and Anosporum plus the segregate genera Courtoisina and Oxycaryum; and clade 3b, C. section Fusci. Clade 4 corresponds to C. section Alternifolii and clade 5 to C. section Leucocephali plus the segregate genus Kyllingiella. The sixth clade is a well‐supported monophyletic clade encompassing all C₄Cyperus s.l. species (‘C₄Cyperus’). This study establishes a phylogenetic framework for future studies. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 19–46.