James R. Manhart

Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the molluscs digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3′ flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty (“green animals”) and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer.

Functional Constraints and rbcL Evidence for Land Plant Phylogeny

Although the proportion of «functional» DNA in eukaryotic genomes is both debatable and subject to definition, most sequences gathered for phylogenetic purposes are indisputably functional. For example, patterns of variation are likely to be strongly constrained in ribosomal RNAs because of their structural and catalytic roles in protein translation, and in protein-coding genes, because of protein function itself. Although seemingly obvious, these concerns are usually ignored by workers producing gene trees. We have examined the extent of functional constraints in land-plant rbcL sequences. Not only do rbcL sequences appear to change with essentially clocklike regularity, but nucleotide-based cladograms imply tbat approximately 97.5% of codon changes on internal branches are functionally neutral (i.e., synonymous or functionally labile)

Phylogeny of the Caryophyllales : Gene sequence data

Phylogenetic relationships among selected families of the Caryophyllales were examined by analyses of nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL). Sequences from 12 Caryophyllalean families were examined using parsimony and bootstrap analysis. The parsimony analysis produced two minimal trees with 778 steps. Major clades supported by bootstrap analysis are (1) Amaranthus (Amaranthaceae), Atriplex and Spinacia (Chenopodiaceae); (2) Mirabilis (Nyctaginaceae), Rivina (Petiveriaceae), Phytolacca (Phytolaccaceae) and Trianthema (Aizoaceae); (3) Basella (Basellaceae), Alluaudia (Didiereaceae), Portulaca (Portulacaceae) and Schlumbergera (Cactaceae); and (4) Silene, Dianthus, Arenaria and Cerastium (Caryophyllaceae). The Caryophyllaceae and Mollugo are the only taxa examined that do not produce betalains but they are neither closely allied nor basal to other elements of the order. Stegnosperma is not allied with the Mirabilis-Rivina-Phytolacca-Trianthema clade. Thus, the Phytolaccaceae (s.l.) are not monophyletic.

Mollusc/algal chloroplast symbiosis: how can isolated chloroplasts continue to function for months in the cytosol of a sea slug in the absence of an algal nucleus?**

A marine sea slug, Elysia chlorotica, has acquired the ability to carry out photosynthesis as a result of forming an intracellular symbiotic association with chloroplasts of the chromophytic alga, Vaucheria litorea. The symbiont chloroplasts (kleptoplasts) are functional, i.e. they evolve oxygen and fix CO(2) and actively transcribe and translate proteins for several months in the sea slug cytosol. Considering the dependency of plastid function on nuclear genes, the level of kleptoplast activity observed in the animal cell is quite remarkable. Possible factors contributing to this long-lasting functional association that are considered here include: the presence of an algal nuclear genome in the sea slug, autonomous chloroplasts, unusual chloroplast/protein stability, re-directing of animal proteins to the kleptoplast, and lateral gene transfer. Based on our current understanding, the acquisition and incorporation of intact algal plastids by E. chlorotica is aided by the robustness of the plastids and the long-term functional activity of the kleptoplasts appears to be supported by both plastid and protein stability and contributions from the sea slug.

Latitudinal variation in cold hardiness in introduced Tamarix and native Populus

To investigate the evolution of clinal variation in an invasive plant, we compared cold hardiness in the introduced saltcedar (Tamarix ramosissima, Tamarix chinensis, and hybrids) and the native plains cottonwood (Populus deltoides subsp. monilifera). In a shadehouse in Colorado (41°N), we grew plants collected along a latitudinal gradient in the central United States (29–48°N). On 17 occasions between September 2005 and June 2006, we determined killing temperatures using freeze‐induced electrolyte leakage and direct observation. In midwinter, cottonwood survived cooling to −70°C, while saltcedar was killed at −33 to −47°C. Frost sensitivity, therefore, may limit northward expansion of saltcedar in North America. Both species demonstrated inherited latitudinal variation in cold hardiness. For example, from September through January killing temperatures for saltcedar from 29.18°N were 5–21°C higher than those for saltcedar from 47.60°N, and on September 26 and October 11, killing temperatures for cottonwood from 33.06°N were >43°C higher than those for cottonwood from 47.60°N. Analysis of nine microsatellite loci showed that southern saltcedars are more closely related to T. chinensis while northern plants are more closely related to T. ramosissima. Hybridization may have introduced the genetic variability necessary for rapid evolution of the cline in saltcedar cold hardiness.

PSEUDO‐NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo‐nitzschia multiseries (Hasle) Hasle and Pseudo‐nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.

Gene Sequence Data

The use of chloroplast DNA comparisons to determine phylogenetic relationships in the angiosperms has become an active area of research. There are three basic types of chloroplast DNA data that have been used for phylogenetic reconstruction: (1) comparison of restriction endonuclease sites (Jansen and Palmer 1988); (2) major genome structural changes (Downie et al. 1991; Downie and Palmer 1992); and (3) comparative gene sequences (Clegg and Zurawski 1992). The first category is covered in Chapter 8. The goal of this chapter is to discuss the use of rbcL sequences to test phylogenetic hypotheses in the Caryophyllales.

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.

UNIQUE CHLOROPLAST GENOME IN SPIROGYRA MAXIMA (CHLOROPHYTA) REVEALED BY PHYSICAL AND GENE MAPPING1

The choroplast genome in Spirogyra maxima (Hassall) Kützing is 130 kb in size and locks large repeat units. The 16S and 23S rRNA genes are separated by approximately 44 kb, which makes co‐transcription of these genes unlikely. Spirogyra maxima is unusual among green algae in that its chloroplast genome apparently does not contain the tufA gene, the absence of which is characteristic of land plant chloroplast DNAs. Apart from three groups of genes, pasA‐psaB, psbC‐psbD, and atpA‐atpF/H, which are co‐transcribed in land plants and also map together in S. maxima chloroplast DNA, the S. maxima gene order overall is unlike that of any other chloroplast genome.

Relationships of Eastern North American Phlox (Polemoniaceae) based on ITS Sequence Data

Phylogenetic relationships of eastern Phlox (Polemoniaceae) are examined using sequence data from the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. Eastern North American Phlox species have been the focus of many evolutionary studies, and hybridization is thought to have played a role in the origin and evolution of some taxa. The phylogenetic information presented here leads to several important conclusions relevant to taxonomy, notably that Wherrys sections Phlox and Aiinnuae are not mono- phyletic, and that the eastern mat-forming taxa and the annuals of central Texas each form monophyletic groups. Neither of the two large species complexes in the east, the P pilosa complex and the P glaberrima complex, is monophyletic in the ITS tree. While the tree is consistent with a history of hybridization, the ITS data do not directly support previous hypotheses of hybrid origin of particular taxa.