Mitochondrial Genome of Prasinophyte Alga Pyramimonas parkeae

Prasinophytes are a paraphyletic assemblage of nine heterogeneous lineages in the Chlorophyta clade of Archaeplastida. Until now, seven complete mitochondrial genomes have been sequenced from four prasinophyte lineages. Here, we report the mitochondrial genome of Pyramimonas parkeae, the first representative of the prasinophyte clade I. The circular‐mapping molecule is 43,294 bp long, AT rich (68.8%), very compact and it comprises two 6,671 bp long inverted repeat regions. The gene content is slightly smaller than the gene‐richest prasinophyte mitochondrial genomes. The single identified intron is located in the cytochrome c oxidase subunit 1 gene (cox1). Interestingly, two exons of cox1 are encoded on the same strand of DNA in the reverse order and the mature mRNA is formed by trans‐splicing. The phylogenetic analysis using the data set of 6,037 positions assembled from 34 mtDNA‐encoded proteins of 48 green algae and plants is not in compliance with the branching order of prasinophyte clades revealed on the basis of 18S rRNA genes and cpDNA‐encoded proteins. However, the phylogenetic analyses based on all three genomic elements support the sister position of prasinophyte clades Pyramimonadales and Mamiellales.     

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

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

Phylogeny of Rhigonematomorpha based on the complete mitochondrial genome of Rhigonema thysanophora (Nematoda: Chromadorea)

We determined the complete mitochondrial genome sequence of Rhigonema thysanophora, the first representative of Rhigonematomorpha, and used this sequence along with 57 other nematode species for phylogenetic analyses. The R. thysanophora mtDNA is 15 015 bp and identical to all other chromadorean nematode mtDNAs published to date in that it contains 36 genes (lacking atp8) encoded in the same direction. Phylogenetic analyses of nucleotide and amino acid sequence data for the 12 protein‐coding genes recovered Rhigonematomorpha as the sister group to the heterakoid species, Ascaridia columbae (Ascaridomorpha). The organization of R. thysanophora mtDNA resembles the most common pattern for the Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha clade in gene order, but with some substantial gene rearrangements. This similarity in gene order is in agreement with the sequence‐based analyses that indicate a close relationship between Rhigonematomorpha and Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha. These results are consistent with certain analyses of nuclear SSU rDNA for R. thysanophora and some earlier classification systems that asserted phylogenetic affinity between Rhigonematomorpha and Ascaridomorpha, but inconsistent with morphology‐based phylogenetic hypotheses that suggested a close (taxonomic) relationship between rhigonematomorphs and oxyuridomorphs (pinworms). These observations must be tempered by noting that few rhigonematomorph species have been sequenced and included in phylogenetic analyses, and preliminary studies based on SSU rDNA suggest the group is not monophyletic. Additional mitochondrial genome sequences of rhigonematids are needed to characterize their phylogenetic relationships within Chromadorea, and to increase understanding of mitochondrial genome evolution.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Occurrence of the gametophyte of Agarum clathratum (Laminariales, Phaeophyceae) as an endophyte in Orculifilum denticulatum (Gigartinales, Rhodophyceae)

An endophytic filamentous brown alga, growing in the red alga Orculifilum denticulatum Lindstrom, was collected from the north‐east Pacific, near Juneau, Alaska. Within the host tissue, its branched filaments formed a network in the space between the filaments of the host tissues embedded in the host intercellular substances. Cells of the filaments contained many discoid chloroplasts without pyrenoids. Neither microscopic morphological observation nor culturing was sufficient to reveal the specific identity or even the familial affinity of the alga; in contrast, molecular phylogenetic analysis of its rbcL gene and rDNA ITS sequences showed that it was the gametophyte of Agarum clathratum Dumortier (Laminariales). There are few reports of laminarialean gametophytes in nature; this is the first report actually identifying the species of laminarialean gametophyte in a red alga.     

Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants

The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non‐seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate‐specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES1

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum‐likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well‐supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile‐haptophyte‐cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.     

Craniid brachiopods: aspects of clade structure and distribution reflect continental drift (Brachiopoda: Craniiformea)

We present maximum likelihood and Bayesian inference relative time‐tree analyses of aligned gene sequences from a worldwide collection of craniiform brachiopods belonging to two genera, Novocrania and Neoancistrocrania. Sequences were obtained from one mitochondrial and three nuclear‐encoded ribosomal RNA genes from varying numbers of specimens. Data‐exploration by network (splits) analyses indicates that each gene identifies the same divergent clades and (with one minor exception) the same inter‐clade relationships. Neoancistrocrania specimens were found only in the Pacific Ocean, near Japan, on the Norfolk and Chesterfield Ridges, and near the Solomon Islands. The Novocrania clades, in approximate order of increasing distance from the root comprise 1. a ‘Northern’ clade of animals collected in the NE. Atlantic, W. Mediterranean and Adriatic; 2. a ‘Tethyan’ clade comprising animals from the E. Mediterranean, Cape Verde islands and the Caribbean (Belize and Jamaica); 3. a ‘NE. Pacific’ clade containing animals from Vancouver Island and from localities near Japan and south of Taiwan; 4. a ‘Southern’ clade that contains two widely separated subclades, one from New Zealand and the other with an extraordinarily wide distribution, ranging from near Japan in the north to the Chesterfield Ridge and Solomon Islands in the West, and in the East to the Galapagos Islands, the coast of South America (Chile) and Richardson seamount (off South Africa) in the South Atlantic. To the South, members of this clade were found in the Weddell, Scotia and Bellinghausen Antarctic Seas. The root of the extant craniid radiation was previously found (by relaxed‐clock analysis) to lie on the branch connecting the two genera so that, in effect, the one clade of Neoancistrocrania serves to polarise evolutionary relationships within the several clades of Novocrania. As previously suggested, all results confirm that Neoancistrocrania is sister to the ‘Northern’ Novocrania clade, and this leads to a proposal that Neoancistrocrania represents one extreme of a wide range of variation in ancestral ventral valve mineralisation, speciation (～90 Ma) resulting from competitive exclusion in rapidly‐growing reef environments. To the extent possible, the identified molecular clades are correlated with named species of Novocrania. The reproductive and population biology of craniid brachiopods is not well known, but from available evidence they are considered to have low‐dispersal potential and, except in enclosed localities such as cold‐water fjords, to have small effective population sizes, features which are consistent with the observed divergent populations in well‐separated localities. Exceptionally slow craniid molecular (rDNA) evolution is suggested by the short branch of Novocrania where it has been used as an outgroup for large‐scale analyses of metazoans. Slow molecular evolution is also indicated by the existence of a distinct Tethyan clade, reflecting restricted dispersal at former times, and by the uniform, short, genetic distances and exceptionally wide geographical distribution of the Southern clade. Thus, the geographical distribution and phylogenetic divergence of craniid brachiopods is an example of phylotectonics, in which relationships revealed by phylogenetic analyses reflect opportunities for dispersal and settlement that were created by tectonic plate movements associated, in this case, with opening and closure of Tethys and the breakup of Gondwana. Molecular dating of craniid divergences and radiochemical dating of tectonic events thus illuminate one another. © 2014 The Linnean Society of London     

GENE SEQUENCE DIVERSITY AND THE PHYLOGENETIC POSITION OF ALGAE ASSIGNED TO THE GENERA PHAEOPHILA AND OCHLOCHAETE (ULVOPHYCEAE,CHLOROPHYTA)1

The phylogenetic position of microfilamentous marine green algae assigned to the species Phaeophila dendroides, Entocladia tenuis (Phaeophila tenuis, and Ochlochaete hystrix was examined through phylogenetic analyses of nuclear‐encoded small subunit rDNA and chloroplast‐encoded tufA gene sequences. These analyses placed the P. dendroides strains within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and the species Bolbocoleon piliferum, supporting an earlier hypothesis that P. dendroides constitutes a distinct lineage. Substantial divergence in both nuclear and plastid DNA sequences exists among strains of P. dendroides from different geographic localities, but these isolated strains are morphologically indistinguishable. The lineage may have an accelerated rate of gene sequence evolution relative to other microfilamentous marine green algae. Entocladia tenuis and O. hystrix are placed neither in the P. dendroides clade nor in the Ulvellaceae as previous taxonomic schemes predicted but instead form a new clade or clades at the base of the Ulvaceae. Ruthnielsenia gen. nov. is proposed to accommodate Kylin's species, which cannot be placed in Entocladia (=Acrochaete), Phaeophila, or Ochlochaete. Ruthnielsenia tenuis (Kylin) comb. nov., previously known only from Atlantic coasts, is reported for the first time from the Pacific coast of North America (San Juan Island, WA, USA). Isolates of R. tenuis from the Atlantic and Pacific coasts of North America have identical small subunit rDNA and tufA gene sequences.     

PHYLOGENETIC RELATIONSHIPS WITHIN CLADOPHORALES (ULVOPHYCEAE,CHLOROPHYTA) INFERRED FROM 18S rRNA GENE SEQUENCES,WITH SPECIAL REFERENCE TO AEGAGROPILA LINNAEI 1

The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

The molecular phylogenetics of Trachymyrmex Forel ants and their fungal cultivars provide insights into the origin and coevolutionary history of ‘higher‐attine’ ant agriculture

The fungus‐growing ants and their fungal cultivars constitute a classic example of a mutualism that has led to complex coevolutionary dynamics spanning c. 55–65 Ma. Of the five agricultural systems practised by fungus‐growing ants, higher‐attine agriculture, of which leaf‐cutter agriculture is a derived subset, remains poorly understood despite its relevance to ecosystem function and human agriculture across the Neotropics and parts of North America. Among the ants practising higher‐attine agriculture, the genus Trachymyrmex Forel, as currently defined, shares most‐recent common ancestors with both the leaf‐cutter ants and the higher‐attine genera Sericomyrmex Mayr and Xerolitor Sosa‐Calvo et al. Although previous molecular‐phylogenetic studies have suggested that Trachymyrmex is a paraphyletic grade, until now insufficient taxon sampling has prevented a full investigation of the evolutionary history of this group and limited the possibility of resolving its taxonomy. Here we describe the results of phylogenetic analyses of 38 Trachymyrmex species, including 27 of the 49 described species and at least 11 new species, using four nuclear markers, as well as phylogenetic analyses of the fungi cultivated by 23 species of Trachymyrmex using two markers. We generated new genetic data for 112 ants (402 new gene sequences) and 95 fungi (153 new gene sequences). Our results corroborate previous findings that Trachymyrmex, as currently defined, is paraphyletic. We propose recognizing two new genera, Mycetomoellerius gen.n. and Paratrachymyrmex gen.n. , and restricting the continued use of Trachymyrmex to the clade of nine largely North American species that contains the type species [Trachymyrmex septentrionalis (McCook)] and that is the sister group of the leaf‐cutting ants. Our fungal cultivar phylogeny generally corroborates previously observed broad patterns of ant–fungus association, but it also reveals further violations of those patterns. Higher‐attine fungi are divided into two groups: (i) the single species Leucoagaricus gongylophorus (Möller); and (ii) its sister clade, consisting of multiple species, recently referred to as Leucoagaricus Singer ‘clade B’. Our phylogeny indicates that, although most non‐leaf‐cutting higher‐attine ants typically cultivate species in clade B, some species cultivate L. gongylophorus, whereas still others cultivate fungi typically associated with lower‐attine agriculture. This indicates that the attine agricultural systems, which are currently defined by associations between ants and fungi, are not entirely congruent with ant and fungal phylogenies. They may, however, be correlated with as yet poorly understood biological traits of the ants and/or of their microbiomes.     

Solenicola setigera is the first characterized member of the abundant and cosmopolitan uncultured marine stramenopile group MAST‐3

Culture‐independent molecular methods based on the amplification, cloning and sequencing of small‐subunit (SSU) rRNA genes are a powerful tool to study the diversity of prokaryotic and eukaryotic microorganisms for which morphological features are not conspicuous. In recent years, molecular data from environmental surveys have revealed several clades of protists lacking cultured and/or described members. Among them are various clades of marine stramenopiles (heterokonts), which are thought to play an essential ecological role as grazers, being abundant and distributed in oceans worldwide. In this work, we show that Solenicola setigera, a distinctive widespread colonial marine protist, is a member of the environmental clade MArine STramenopile 3 (MAST‐3). Solenicola is generally considered as a parasite or an epiphyte of the diatom Leptocylindrus mediterraneus. So far, the ultrastructural, morphological and ecological data available were insufficient to elucidate its phylogenetic position, even at the division or class level. We determined SSU rRNA gene sequences of S. setigera specimens sampled from different locations and seasons in the type locality, the Gulf of Lions, France. They were closely related, though not identical, which, together with morphological differences under electron microscopy, suggest the occurrence of several species. Solenicola sequences were well nested within the MAST‐3 clade in phylogenetic trees. Since Solenicola is the first identified member of this abundant marine clade, we propose the name Solenicolida for the MAST‐3 phylogenetic group.     

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae,Chlamydomonadales)

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.     

Phylogeography of the pallid kangaroo mouse,Microdipodops pallidus: a sand‐obligate endemic of the Great Basin,western North America

Aim Kangaroo mice, genus Microdipodops Merriam, are endemic to the Great Basin and include two species: M. pallidus Merriam and M. megacephalus Merriam. The pallid kangaroo mouse, M. pallidus, is a sand‐obligate desert rodent. Our principal intent is to identify its current geographical distribution and to formulate a phylogeographical hypothesis for this taxon. In addition, we test for orientation patterns in haplotype sharing for evidence of past episodes of movement and gene flow. Location The Great Basin Desert region of western North America, especially the sandy habitats of the Lahontan Trough and those in south‐central Nevada. Methods Mitochondrial DNA sequence data from portions of three genes (16S ribosomal RNA, cytochrome b, and transfer RNA for glutamic acid) were obtained from 98 individuals of M. pallidus representing 27 general localities sampled throughout its geographical range. Molecular sequence data were analysed using neighbour‐joining, maximum‐parsimony, maximum‐likelihood and Bayesian methods of phylogenetic inference. Directional analysis of phylogeographical patterns, a novel method, was used to examine angular measurements of haplotype sharing between pairs of localities to detect and quantify historical events pertaining to movement patterns and gene flow. Results Collecting activities showed that M. pallidus is a rather rare rodent (mean trapping success was 2.88%), and its distribution has changed little from that determined three‐quarters of a century ago. Two principal phylogroups, distributed as eastern and western moieties, are evident from the phylogenetic analyses (mean sequence divergence for cytochrome b is c. 8%). The western clade shows little phylogenetic structure and seems to represent a large polytomy. In the eastern clade, however, three subgroups are recognized. Nine of the 42 unique composite haplotypes are present at two or more localities and are used for the orientation analyses. Axial data from haplotype sharing between pairwise localities show significant, non‐random angular patterns: a north‐west to south‐east orientation in the western clade, and a north‐east to south‐west directional pattern in the eastern clade. Main conclusions The geographical range of M. pallidus seems to be remarkably stable in historical times and does not show a northward (or elevationally upward) movement trend, as has been reported for some other kinds of organism in response to global climate change. The eastern and western clades are likely to represent morphologically cryptic species. Estimated times of divergence of the principal clades of M. pallidus (4.38 Ma) and between M. pallidus and M. megacephalus (8.1 Ma; data from a related study) indicate that kangaroo mice diverged much earlier than thought previously. The phylogeographical patterns described here may serve as a model for other sand‐obligate members of the Great Basin Desert biota.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

PHYLOGENETIC ANALYSES OF THE BRYOPSIDALES (ULVOPHYCEAE,CHLOROPHYTA) BASED ON RUBISCO LARGE SUBUNIT GENE SEQUENCES1

Current taxonomy of the Bryopsidales recognizes eight families; most of which are further categorized into two suborders, the Bryopsidineae and Halimedineae. This concept was supported by early molecular phylogenetic analyses based on rRNA sequence data, but subsequent cladistic analyses of morphological characters inferred monophyly in only the Halimedineae. These conflicting results prompted the current analysis of 32 taxa from this diverse group of green algae based on plastid‐encoded RUBISCO large subunit (rbcL) gene sequences. Results of these analyses suggested that the Halimedineae and Bryopsidineae are distinct monophyletic lineages. The families Bryopsidaceae, Caulerpaceae, Codiaceae, Derbesiaceae, and Halimediaceae were inferred as monophyletic, however the Udoteaceae was inferred as non‐monophyletic. The phylogenetic position of two taxa with uncertain subordinal affinity, Dichotomosiphon tuberosus Lawson and Pseudocodium floridanum Dawes & Mathieson, were also inferred. Pseudocodium was consistently placed within the halimedinean clade suggesting its inclusion into this suborder, however familial affinity was not resolved. D. tuberosus was the inferred sister taxon of the Halimedineae based on analyses of rbcL sequence data and thus a possible member of this suborder.     

PHYLOGENETIC POSITION OF BOLBOCOLEON PILIFERUM (ULVOPHYCEAE,CHLOROPHYTA): EVIDENCE FROM REPRODUCTION,ZOOSPORE AND GAMETE ULTRASTRUCTURE,AND SMALL SUBUNIT RRNA GENE SEQUENCES1

Aspects of the reproduction of Bolbocoleon piliferum N. Pringsheim, a common, small, filamentous, endophytic marine green alga, were examined by LM and TEM. These observations were combined with phylogenetic analysis of nuclear‐encoded small subunit rRNA gene sequences to assess the phylogenetic position of B. piliferum. Quadriflagellate zoospores and planozygotes derived from fusion of isogametes yielded plants with identical morphology. Zoosporangia and gametangia divided by sequential cleavages. Plugs at the apices of zoosporangia and gametangia formed during development; tubes were found at zoosporangial and gametangial apices after swarmer release. Flagellar apparatuses of zoospores and gametes were similar to those of algae in the Ulvales (Ulvophyceae), except that terminal caps were entire rather than bilobed and rhizoplasts and “stacked” microtubular root configurations were absent. Structures associated with planozygotes were identical to those observed in other algae currently assigned to Ulotrichales and Ulvales. Molecular phylogenetic analyses placed B. piliferum within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and Kornmanniaceae. The results support an earlier hypothesis that B. piliferum constitutes a distinct lineage. Analyses including Kornmanniaceae recover monophyletic Ulotrichales and Ulvales, whereas analyses omitting the Kornmanniaceae indicate that Ulotrichales is paraphyletic. The structures associated with gamete fusion are conserved within Ulotrichales and Ulvales and perhaps more widely within Chlorophyta.