Minimum Evolution Analyses Research Articles

Geography shapes the phylogeny of frailejones (Espeletiinae Cuatrec., Asteraceae): a remarkable example of recent rapid radiation in sky islands.

BackgroundThe páramo ecosystem, located above the timberline in the tropical Andes, has been the setting for some of the most dramatic plant radiations, and it is one of the world’s fastest evolving and most diverse high-altitude ecosystems. Today 144+ species of frailejones (subtribe Espeletiinae Cuatrec., Asteraceae) dominate the páramo. Frailejones have intrigued naturalists and botanists, not just for their appealing beauty and impressive morphological diversity, but also for their remarkable adaptations to the extremely harsh environmental conditions of the páramo. Previous attempts to reconstruct the evolutionary history of this group failed to resolve relationships among genera and species, and there is no agreement regarding the classification of the group. Thus, our goal was to reconstruct the phylogeny of the frailejones and to test the influence of the geography on it as a first step to understanding the patterns of radiation of these plants.MethodsField expeditions in 70 páramos of Colombia and Venezuela resulted in 555 collected samples from 110 species. Additional material was obtained from herbarium specimens. Sequence data included nrDNA (ITS and ETS) and cpDNA (rpl16), for an aligned total of 2,954 bp. Fragment analysis was performed with AFLP data using 28 primer combinations and yielding 1,665 fragments. Phylogenies based on sequence data were reconstructed under maximum parsimony, maximum likelihood and Bayesian inference. The AFLP dataset employed minimum evolution analyses. A Monte Carlo permutation test was used to infer the influence of the geography on the phylogeny.ResultsPhylogenies reconstructed suggest that most genera are paraphyletic, but the phylogenetic signal may be misled by hybridization and incomplete lineage sorting. A tree with all the available molecular data shows two large clades: one of primarily Venezuelan species that includes a few neighboring Colombian species; and a second clade of only Colombian species. Results from the Monte Carlo permutation test suggests a very strong influence of the geography on the phylogenetic relationships. Venezuelan páramos tend to hold taxa that are more distantly-related to each other than Colombian páramos, where taxa are more closely-related to each other.ConclusionsOur data suggest the presence of two independent radiations: one in Venezuela and the other in Colombia. In addition, the current generic classification will need to be deeply revised. Analyses show a strong geographic structure in the phylogeny, with large clades grouped in hotspots of diversity at a regional scale, and in páramo localities at a local scale. Differences in the degrees of relatedness between sympatric species of Venezuelan and Colombian páramos may be explained because of the younger age of the latter páramos, and the lesser time for speciation of Espeletiinae in them.

SRAP Markers Based Genetic Analysis of Silene Species

Plant genetic resources are an essential part of the world’s natural resources, and knowledge of genetic diversity and its distribution is essential for plant conservation. In the present work, the genetic variation was studied among thirteen species of Silene genus. Samples were collected from different locations of West Azarbijan and Khorasan provinces in Iran. This investigation, for the first time, has studied sequence-related amplified polymorphism (SRAP) marker to assess the genetic diversity and genetic similarity among 13 species of Silene in Iran. These species belonge to eight sections including Sclerocalycinae, Melandriforms, Lychnidiforms, Inflatae, Lasiocalycinae, Spergolifoliae, Auriculatae and Conoimorpha. Fifteen SRAP primer combinations generated 62 fragments, of which 46 (71.90 %) were polymorphic. Percentage of polymorphism ranged from 50 % to a maximum of 100 % and Jaccard’s similarity coefficient ranged from 0.48 to 0.91. Minimum Evolution analysis revealed four main clusters. The study indicates that SRAP markers with targeting ORFs, high reproducibility and optimal marker distribution could be good candidates for assessing genetic variation in Silene.

Genetic structure and phylogeny of Italian and Czech populations of the cucurbit powdery mildew fungus Golovinomyces orontii inferred by multilocus sequence typing

Cucurbit powdery mildew caused by Golovinomyces orontii is a serious disease that affects cucurbit crops in temperate areas. In northern Italy, the species is responsible for the early infections at the beginning of the growing season. However, chasmothecia have never been recorded in Italy and the impact of either asexual or sexual reproduction of G. orontii remains to be determined. To investigate and compare the genetic structure of Italian populations with those from the Czech Republic, where chasmothecia are sporadically recorded, seven housekeeping gene fragments (tef‐1α, csI, ITS, H3, tub2, IGS and mtLSU) were amplified and used in a multilocus sequence typing (MLST) approach. The four‐gamete test was performed to detect recombination between and within populations. Phylogeny was inferred using both Bayesian and minimum evolution analyses. Results from MLST revealed the presence of 141 single nucleotide polymorphisms and the existence of 13 different haplotypes. Phylogenetic analysis revealed the existence of two main distinct phylogenetic groups that were also highly genetically different, confirming that G. orontii is a species complex. Recombination was detected within both the phylogenetic groups and the geographical populations, indicating that sexual reproduction could have occurred. However, considering the lower haplotype diversity and the high frequency of ST3 (17 isolates) and ST7 (eight isolates) haplotypes, the Italian population could be predominantly clonal, while sexual reproduction may occasionally occur due to the introduction of genotypes similar to those from the Czech Republic.

Molecular Systematics of Tanaidacea (Crustacea: Peracarida) Based on 18S Sequence Data, with an Amendment of Suborder/Superfamily-Level Classification

Phylogenetic relationships within Tanaidacea were analyzed based on sequence data for the 18S rRNA gene. Our results strongly supported a monophyletic group composed of Neotanaidae, Tanaoidea, and Paratanaoidea, with the first two taxa forming a clade. These results contradict three previously suggested hypotheses of relationships. Based on the molecular results, and considering morphological similarities/differences between Neotanaidomorpha and Tanaidomorpha, we demoted Suborder Neotanaidomorpha to Superfamily Neotanaoidea within Tanaidomorpha; with this change, the classification of extant tanaidaceans becomes a two-suborder, four-superfamily system. This revision required revision of the diagnoses for Tanaidomorpha and its three super-families. The results for Apseudomorpha were ambiguous: this taxon was monophyletic in the maximum likelihood and Bayesian analyses, but paraphyletic in the maximum parsimony and minimum evolution analyses.

Complete sequence and organisation of the Jatropha curcas (Euphorbiaceae) chloroplast genome

Jatropha curcas is an important non-edible oil seed tree species and is considered a promising source of biodiesel. The complete nucleotide sequence of J. curcas chloroplast genome (cpDNA) was determined by pyrosequencing and gaps filled by Sanger sequencing. The cpDNA is a circular molecule of 163,856 bp in length and codes for 110 distinct genes (78 protein coding, four rRNA and 28 distinct tRNA). Genome organisation and arrangement are similar to the reported angiosperm chloroplast genome. However, in Jatropha, the infA and the rps16 genes are non-functional. The inverted repeat (IR) boundary is within the rpl2 gene, and the 13 nucleotides at the ends of the two duplicate genes are different. Repeat analysis suggests the presence of 72 repeat regions (>30 bp) apart from the IR; of these, 48 were direct and 24 were palindromic repeats. Phylogenetic analysis of 81 protein coding chloroplast genes from 65 taxa by maximum parsimony, maximum likelihood and minimum evolution analyses at 100 bootstraps provide strong support for the placement of inaperturate crotonoids of which Jatropha is a member as sister to articulated crotonoids of which Manihot is a member.

A Phylogeny of the Genus Polemonium Based on Amplified Fragment Length Polymorphism (AFLP) Markers

The genus Polemonium is composed of animal-pollinated herbs found largely in North America. Wide phenotypic variation suggests rapid evolution of floral and vegetative morphology. However, phylogenetic relationships within Polemonium have received little attention. We present a phylogeny of Polemonium based on 1676 Amplified Fragment Length Polymorphisms (AFLPs). We obtained similar topologies through minimum evolution analysis using Nei-Li genetic distances and parsimony analysis. Our analyses supported the monophyly of most species, and indicated that the single annual species, P. micranthum, is sister to all perennial species. Relationships were well resolved among species separated by genetic distances < 0.16. Three well-supported clades each included taxa from one of three species complexes: the P. caeruleum complex, the P. pulcherrimum complex, or the P. viscosum complex. Relationships within clades were generally consistent with earlier systematic work. Exceptions were the inclusion of eastern North American P. reptans within the P. caeruleum clade, inclusion of P. boreale within the P. pulcherrimum clade, and a close relationship between tetraploid P. californicum and diploid P. pulcherrimum subsp. delicatum. Our results indicate that floral traits are quite divergent among close relatives within the genus, and provide a phylogenetic framework for research on adaptation and speciation in Polemonium.

Morphological disparity despite genetic similarity; new species of Lobosorchis Miller & Cribb, 2005 (Digenea: Cryptogonimidae) from the Great Barrier Reef and the Maldives

Examination of the humpback red snapper, Lutjanus gibbus (Perciformes: Lutjanidae), from Lizard Island off the Great Barrier Reef and Rasdhoo Atoll, Maldives revealed the presence of a new species of Lobosorchis (Digenea: Cryptogonimidae), L. polygongylus n. sp. Lobosorchis polygongylus n. sp. is distinguished from the typeand only other species, L. tibaldiae by the combination of body size, oral spine number (60–81 in L. polygongylus, 47–56 in L. tibaldiae) and number of testes (13–25 in L. polygongylus, 9 in L. tibaldiae). Bayesian inference analysis using data from the internal transcribed spacers 1 and 2 (ITS1 and ITS2), 5.8S and the large subunit (LSU) nuclear ribosomal DNA of L. polygongylus, L. tibaldiae and species of Beluesca, Caulanus, Chelediadema, Neometadena, Latuterus, Retrovarium and Siphoderina was performed to explore phylogenetic relationships of species of Lobosorchis with other cryptogonimid taxa. Despite the significant morphological differences between Lobosorchis polygongylus and L. tibaldiae, these two species differed consistently by only 5 base pairs (bp) over the entire ITS region (3 bp in ITS1, 0 bp in 5.8S and 2 bp in ITS2) and 1 bp in the LSU rDNA regions examined. The ITS2 rDNA region was sequenced from metacercariae obtained from the fins, flesh or body cavities of a number of fishes belonging to the Blenniidae, Pomacentridae and Tetraodontidae and analysed using minimum evolution analysis with L. polygongylus and L. tibaldiae. This revealed the presence of two additional genotypes (putative Lobosorchis sp. A and B), which consistently differed from L. polygongylus by 1 and 4 bp, L. tibaldiae by 1 and 4 bp and from each other by 3 bp over the ITS2 dataset. Although these genetic differences are relatively small, when evaluated in light of the differences observed between L. polygongylus and L. tibaldiae (which are morphologically quite distinct) and differences seen in other congeneric cryptogonimid taxa, the ITS2 rDNA data alone suggest that at least two more species of Lobosorchis are present at Lizard Island. These data also suggest that the ITS2 rDNA region alone is suitable for resolving operational taxonomic units (OTUs) at the species level within the Cryptogonimidae based on what was observed in this and other cryptogonimid systems. A morphological description of metacercariae of L. tibaldiae obtained from two species of Pomacentridae at Heron Island, off the Great Barrier Reef is also provided.

Phylogenetic Relationships of Basal Hexapods Reconstructed from Nearly Complete 18S and 28S rRNA Gene Sequences

This study combined nearly complete 28S and 18S rRNA gene sequences (>4100 nt long) to investigate the phylogenetic relationships of basal hexapods (Protura, Collembola, and Diplura). It sequenced more 28S genes, to expand on a previous study from this lab that used 18S plus only a tiny part of the 28S gene. Sixteen species of basal hexapods, five insects, six crustaceans, two myriapods, and two chelicerates were included in the analyses. Trees were constructed with maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded consistent results: (1) Hexapoda was monophyletic and nested in a paraphyletic Crustacea, and Hexapoda was divided into Entognatha [Collembola+Nonoculata (Protura plus Diplura)] and Insecta (=Ectognatha), but the Nonoculata clade must be accepted with caution because of its strong nonstationarity of nucleotide composition. (2) Within Diplura, the monophyly of Campodeoidea and of Japygoidea were supported respectively, and all methods united Projapygoidea with Japygoidea. (3) Within Protura, Sinentomidae was the sister group to Acerentomata. (4) Within Collembola, the modern taxonomical hierarchy of Collembola (Poduromorpha, Entomobryomorpha, Symphypleona and Neelipleona) was confirmed.

Two new cryptogonimid genera Beluesca n. gen. and Chelediadema n. gen. (Digenea: Cryptogonimidae) from tropical Indo-West Pacific Haemulidae (Perciformes)

A survey of the parasites of Indo-West Pacific Haemulidae revealed the presence of three new cryptogonimid (Digenea: Cryptogonimidae) species warranting two new genera, Beluesca littlewoodi n. gen., n. sp. and B. longicolla n. sp. from the intestine and pyloric caeca of Plectorhinchus gibbosus and Chelediadema marjoriae n. gen., n. sp. from the intestine and pyloric caeca of Diagramma labiosum, P. albovittatus and P. gibbosus from Heron and Lizard Islands off the Great Barrier Reef, Australia. Beluesca n. gen. is distinguished from all other cryptogonimid genera by the combination of an elongate body, funnel-shaped oral sucker, relatively small number of large oral spines, highly lobed ovary, opposite to slightly oblique testes, uterine loops that are restricted to the hindbody and extend well posterior to the testes and vitelline follicles that may extend from the ovary into the forebody, but do not extend anterior to the intestinal bifurcation. Pseudallacanthochasmus plectorhynchi Mamaev, 1970 is transferred to Beluesca as B. plectorhyncha (Mamaev, 1970) n. comb. based on morphological and ecological (host preference) characteristics. Chelediadema n. gen. is distinguished from all other cryptogonimid genera by the combination of a lanceolate body, relatively small number of large oral spines, prepharynx that is much longer than the oesophagus, tandem testes, uterine loops that are extensive in the hindbody and extend well posterior to the testes and vitelline follicles that extend from the ovary to the pharynx. Morphological analysis of the three species described here was augmented with DNA sequence analyses utilizing data from the large subunit (LSU) and the internal transcribed spacers (ITS) 1 and 2, and 5.8S nuclear ribosomal DNA. Sequence data from the LSU and ITS (encompassing the ITS1, 5.8S and ITS2) of the taxa examined here were aligned with those reported for other cryptogonimids, Caulanus thomasi, Latuterus tkachi, Neometadena ovata, four representative species of Retrovarium and an undescribed species of Siphoderina, for comparative purposes and to explore levels of interspecific and intergeneric variation among these taxa. Minimum evolution analysis was conducted on a combined (LSU and ITS) dataset to explore relationships among these genera. Despite their superficial morphological and host preference similarities, species of Beluesca and Chelediadema were genetically distant from each other. Interspecific and intergeneric variation among the species described here is similar to that reported for other cryptogonimids.

Resolving genetic relationships in manna group of lichens from genus Aspicilia

As in many lichen-forming fungi, species of Aspicilia genus are widely distributed all over the world, but no reports exist about their phylogenetic relations based on molecular biological methods. In the current study the phylogenetic relations of some Aspicilia species mainly manna group of lichens were investigated. The ITS rDNA sequence information of 12 samples from six species were generated. The samples examined were collected from different provinces of Anatolia and all the sequences were aligned with the other allied groups; Pertusaria sp., Thamnolia sp., Dibaeis sp., Diploschistes sp., Ochrolechia sp. and Lecanora sp. sequence data obtained from GenBank. The phylogenetic tree obtained by minimum evolution analysis displayed two major branches. One of the branches with its six members (Aspicilia contorta subsp. contorta, A. contorta subsp. hoffmanniana, Aspicilia hispida, Aspicilia fruticulosa, Aspicilia desertorum Aspicilia calcarea) composed solely of Aspicilia samples from Anatolia. Three of the manna groups of lichens were placed in this branch of the tree. The other related taxa from Ostropomycetidae and Lecanoromycetidae took place in the other branch of the tree with Aspicilia samples from Anatolia. Results displayed that manna group of lichens, although do not represent taxonomical unit tend to form a group especially apperent by the Neigbour Joining analysis with Anatolian samples. Also the sequence information from Anatolian samples displayed that Aspicilia genus is phylogenetically closer to the orders and families from Ostropomycetidae subclass rather than Lecanoromycetidae which support the placement of Hymeneliaceae within Ostropomycetidae.

Coevolution of Retrovarium n. gen. (Digenea: Cryptogonimidae) in Lutjanidae and Haemulidae (Perciformes) in the Indo-West Pacific

We describe 11 new species of cryptogonimids belonging to Retrovarium n. gen., from eight species of Lutjanidae and one species of Haemulidae, from the Great Barrier Reef, French Polynesia and the Maldives. We also transfer Neoparacryptogonimus saccatus (Manter, 1963) and Neoparacryptogonimus sphericus Nahhas, Sey & Nishimoto, 1998 to Retrovarium. The morphologically based taxonomic approach was augmented with DNA sequence data from three nuclear ribosomal DNA regions (28S, ITS1 and ITS2) to explore the species integrity, biogeographic distribution and evolution of the species recognised here. Sequencing included multiple replicates and revealed 11 distinct genotypes which corroborated our morphologically based hypotheses of putative species present in the system. There was no intraspecific variation and all three rDNA regions differed between every combination of species. Two species exhibited wide geographic ranges, having identical rDNA sequences between the Great Barrier Reef and the Maldives, localities separated by over 9600km. One host species, Symphorus nematophorus, proved to be exceptionally rich, harbouring six species. Minimum evolution analyses were conducted on each of the rDNA datasets independently; minimum evolution, maximum likelihood and Bayesian inference analyses were conducted on a combined (28S, ITS1 and ITS2) dataset for sequence comparison purposes and to explore the evolutionary history of these parasites. To examine the coevolutionary history of this complex, assessment of phylogenetic relationships between the 23 species of Lutjanidae and two species of Haemulidae collected during this survey was performed with data from 16S and cytochrome b mtDNA using Bayesian inference analysis. Despite the high host specificity observed in most of the species, mapping of the parasites on the host phylogeny revealed an absence of strict coevolution or co-descent within this complex. Overall, Retrovarium appears to have had an exceptionally patchy radiation, failing to infect many taxa, infecting species with no readily discernible pattern, and radiating dramatically within one species.

Resolving genetic relationships in manna group of lichens from genus Aspicilia

As in many lichen-forming fungi, species of Aspicilia genus are widely distributed all over the world, but no reports exist about their phylogenetic relations based on molecular biological methods. In the current study the phylogenetic relations of some Aspicilia species mainly manna group of lichens were investigated. The ITS rDNA sequence information of 12 samples from six species were generated. The samples examined were collected from different provinces of Anatolia and all the sequences were aligned with the other allied groups; Pertusaria sp., Thamnolia sp., Dibaeis sp., Diploschistes sp., Ochrolechia sp. and Lecanora sp. sequence data obtained from GenBank. The phylogenetic tree obtained by minimum evolution analysis displayed two major branches. One of the branches with its six members (Aspicilia contorta subsp. contorta, A. contorta subsp. hoffmanniana, Aspicilia hispida, Aspicilia fruticulosa, Aspicilia desertorum Aspicilia calcarea) composed solely of Aspicilia samples from Anatolia. Three of the manna groups of lichens were placed in this branch of the tree. The other related taxa from Ostropomycetidae and Lecanoromycetidae took place in the other branch of the tree with Aspicilia samples from Anatolia. Results displayed that manna group of lichens, although do not represent taxonomical unit tend to form a group especially apperent by the Neigbour Joining analysis with Anatolian samples. Also the sequence information from Anatolian samples displayed that Aspicilia genus is phylogenetically closer to the orders and families from Ostropomycetidae subclass rather than Lecanoromycetidae which support the placement of Hymeneliaceae within Ostropomycetidae.

Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

BackgroundLeptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing.ResultsA conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results.ConclusionThis research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.

The Phylogenetic Positions of Three Basal-Hexapod Groups (Protura, Diplura, and Collembola) Based on Ribosomal RNA Gene Sequences

This study combined complete 18S with partial 28S ribosomal RNA gene sequences ( approximately 2,000 nt in total) to investigate the relations of basal hexapods. Ten species of Protura, 12 of Diplura, and 10 of Collembola (representing all subgroups of these three clades) were sequenced, along with 5 true insects and 8 other arthropods, which served as out-groups. Trees were constructed with maximum parsimony, maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded strong support for a clade of Protura plus Diplura, here named Nonoculata, and for monophyly of the Diplura. Parametric-bootstrapping analysis showed our data to be inconsistent with previous hypotheses (P < 0.01) that joined Protura with Collembola (Ellipura), that said Diplura are sister to true insects or are diphyletic, and that said Collembola are not hexapods. That is, our data are consistent with hexapod monophyly and Collembola grouped weakly with "Protura + Diplura" under most analytical conditions. As a caveat to the above conclusions, the sequences showed nonstationarity of nucleotide frequencies across taxa, so the CG-rich sequences of the diplurans and proturans may have grouped together artifactually; however, the fact that the LogDet method supported this group lessens this possibility. Within the basal hexapod groups, where nucleotide frequencies were stationary, traditional taxonomic subgroups generally were recovered: i.e., within Protura, the Eosentomata and Acerentomata (but Sinentomata was not monophyletic); within Collembola, the Arthropleona, Poduromorpha, and Entomobryomorpha (but Symphypleona was polyphyletic); and in Diplura, the most complete data set (> 2,100 nt) showed monophyly of Campodeoidea and of Japygoidea, and most methods united Projapygoidea with Japygoidea.

SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES, RHODOPHYTA): A CRITICAL ASSESSMENT BASED ON RBCL SEQUENCE ANALYSES1

Generic concepts in the economically important agarophyte red algal family Gracilariaceae were evaluated based on maximum parsimony, Bayesian likelihood, and minimum evolution analyses of the chloroplast‐encoded rbc L gene from 67 specimens worldwide. The results confirm the monophyly of the family and identify three large clades, one of which corresponds to the ancestral antiboreal genera Curdiea and Melanthalia, one to Gracilariopsis, and one to Gracilaria sensu lato, which contains nine distinct independent evolutionary lineages, including Hydropuntia. The species currently attributed to Hydropuntia comprise a single well‐supported clade composed of two distinct lineages. The two most basal clades within Gracilaria sensu lato deserve generic rank: a new genus centered around G. chilensis Bird, McLachlan et Oliveira and G. aff. tenuistipitata Chang et Xia and a resurrected Hydropuntia encompassing primarily Indo‐Pacific (G. urvillei [Montagne] Abbott, G. edulis [S. Gmelin] P. Silva, G. eucheumatoides Harvey, G. preissiana [Sonder] Womersley, and G. rangiferina [Kützing] Piccone) and western Atlantic species (G. cornea J. Agardh, G. crassissima P. et H. Crouan in Mazé et Schramm, G. usneoides [C. Agardh] J. Agardh, G. caudata J. Agardh, and G. secunda P. et H. Crouan in Mazé et Schramm). Cystocarpic features within the Gracilaria sensu lato clades appear to be more phylogenetically informative than male characters. The textorii‐type spermatangial configuration is represented in two distinct clusters of Gracilaria. The rbc L genetic divergence among the Gracilariaceae genera ranged between 8.46% and 16.41%, providing at least 2.5 times more genetic variation than does the 18S nuclear rDNA. rbc L also resolves intrageneric relationships, especially within Gracilaria sensu lato. The current number of gracilariacean species is underestimated in the western Atlantic because of convergence in habit and apparent homoplasy in vegetative and reproductive anatomy.

Phylogenetic relationships and genotyping of the genus Streptococcus by sequence determination of the RNase P RNA gene, rnpB.

The rnpB gene is universally present in bacterial species and encodes the RNA subunit of endoribonuclease P. In this study, rnpB was sequenced in 50 type strains and 29 additional strains of the genus Streptococcus. Putative secondary-structure models and possible interactions in RNase P RNA molecules are discussed. Phylogenetic relationships were studied and Bayesian, maximum-parsimony and minimum-evolution analyses supported six main clades that comprised 22 of the 50 species analysed. Phylogenetic inference was also studied for the 16S rRNA gene; it indicated a similar tree topology, but with weaker support values than for rnpB. Combined analysis of rnpB and 16S resulted in a phylogeny with significantly better support. Variability in the rnpB and 16S genes among all type strains, calculated as Shannon-Wiener information index values, was 0.45 for rnpB and 0.15 for 16S. Intraspecies proximity was assessed by principal coordinate analysis of rnpB for 32 strains of six closely related species (two clades) and showed species-specific clusters, but heterogeneity occurred in two species. It can be concluded that the rnpB gene is suitable for phylogenetic analysis of closely related taxa and has potential as a tool for species discrimination.

Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin

Relationships among the ecdysozoans, or molting animals, have been difficult to resolve. Here, we use nearly complete 28S+18S ribosomal RNA gene sequences to estimate the relations of 35 ecdysozoan taxa, including newly obtained 28S sequences from 25 of these. The tree-building algorithms were likelihood-based Bayesian inference and minimum-evolution analysis of LogDet-transformed distances, and hypotheses were tested wth parametric bootstrapping. Better taxonomic resolution and recovery of established taxa were obtained here, especially with Bayesian inference, than in previous parsimony-based studies that used 18S rRNA sequences (or 18S plus small parts of 28S). In our gene trees, priapulan worms represent the basal ecdysozoans, followed by nematomorphs, or nematomorphs plus nematodes, followed by Panarthropoda. Panarthropoda was monophyletic with high support, although the relationships among its three phyla (arthropods, onychophorans, tardigrades) remain uncertain. The four groups of arthropods—hexapods (insects and related forms), crustaceans, chelicerates (spiders, scorpions, horseshoe crabs), and myriapods (centipedes, millipedes, and relatives)—formed two well-supported clades: Hexapoda in a paraphyletic crustacea (Pancrustacea), and `Chelicerata+Myriapoda' (a clade that we name `Paradoxopoda'). Pycnogonids (sea spiders) were either chelicerates or part of the `chelicerate+myriapod' clade, but not basal arthropods. Certain clades derived from morphological taxonomy, such as Mandibulata, Atelocerata, Schizoramia, Maxillopoda and Cycloneuralia, are inconsistent with these rRNA data. The 28S gene contained more signal than the 18S gene, and contributed to the improved phylogenetic resolution. Our findings are similar to those obtained from mitochondrial and nuclear (e.g., elongation factor, RNA polymerase, Hox) protein-encoding genes, and should revive interest in using rRNA genes to study arthropod and ecdysozoan relationships.

Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes.

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.

Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences.

The phylogenetic relationships of thirteen Diabrotica (representing virgifera and fucata species groups) and two outgroup Acalymma beetle species (Coleoptera: Chrysomelidae) were inferred from the phylogenetic analysis of a combined data set of 1323 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit 1 (COI) and the entire second internal transcribed spacer region (ITS-2) of nuclear ribosomal DNA of 362 characters. Species investigated were D. adelpha, D. balteata, D. barberi, D. cristata, D. lemniscata, D. longicornis, D. porracea, D. speciosa, D. undecimpunctata howardi, D. u. undecimpunctata, D. virgifera virgifera, D. v. zeae, D. viridula, and outgroup A. blandulum and A. vittatum. Maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) analyses of combined COI and ITS-2 sequences clearly place species into their traditional morphological species groups with MP and ME analyses resulting in identical topologies. Results generally confer with a prior work based on allozyme data, but within the virgifera species group, D. barberi and D. longicornis strongly resolve as sister taxa as well as monophyletic with the neotropical species, D. viridula, D. cristata and D. lemniscata also resolve as sister taxa. Both relationships are not in congruence with the prior allozyme-based hypothesis. Within the fucata species group, D. speciosa and D. balteata resolve as sister taxa. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. Our proposed phylogeny provides some insight into current hypotheses regarding distribution status and evolution of various life history traits for Diabrotica.

Interrelationships and Evolution of the Tapeworms (Platyhelminthes: Cestoda)

Interrelationships of the tapeworms (Platyhelminthes: Cestoda) were examined by use of small (SSU) and large (LSU) subunit ribosomal DNA sequences and morphological characters. Fifty new complete SSU sequences were added to 21 sequences previously determined, and 71 new LSU (D1–D3) sequences were determined for the complementary set of taxa representing each of the major lineages of cestodes as currently understood. New sequences were determined for three amphilinidean taxa, but were removed from both alignments due to their excessively high degree of divergence from other cestode sequences. A morphological character matrix coded for supraspecific taxa was constructed by the modification of matrices from recently published studies. Maximum-parsimony (MP) analyses were performed on the LSU, SSU, LSU+SSU, and morphological data partitions, and minimum-evolution (ME) analyses utilizing a general time reversible model of nucleotide substitution including estimates of among-site rate heterogeneity were performed on the molecular data partitions. Resulting topologies were rooted at the node separating the Gyrocotylidea from the Eucestoda. The LSU data were found to be more informative than the SSU data and were more consistent with inferences from morphology, although nodal support was generally weak for most basal nodes. One class of transitions was found to be saturated for comparisons between the most distantly related taxa (gyrocotylideans vs cyclophyllideans and tetrabothriideans). Differences in the topologies resulting from MP and ME analyses were not statistically significant. Nonstrobilate orders formed the basal lineages of trees resulting from analysis of LSU data and morphology. Difossate orders were basal to tetrafossate orders, the latter of which formed a strongly supported clade. A clade including the orders Cyclophyllidea, Nippotaeniidea, and Tetrabothriidea was supported by all data partitions and methods of analysis. Paraphyly of the orders Pseudophyllidea, Tetraphyllidea, and Trypanorhyncha was consistent among the molecular data partitions. Inferences are made regarding a monozoic (nonsegmented) origin of the Eucestoda as represented by the Caryophyllidea and for the evolution of the strobilate and acetabulate/tetrafossate conditions having evolved in a stepwise pattern.