Sources of signal in 62 protein-coding nuclear genes for higher-level phylogenetics of arthropods

Background

This study aims to investigate the strength of various sources of phylogenetic information that led to recent seemingly robust conclusions about higher-level arthropod phylogeny and to assess the role of excluding or downweighting synonymous change for arriving at those conclusions.

Methodology/Principal Findings

The current study analyzes DNA sequences from 68 gene segments of 62 distinct protein-coding nuclear genes for 80 species. Gene segments analyzed individually support numerous nodes recovered in combined-gene analyses, but few of the higher-level nodes of greatest current interest. However, neither is there support for conflicting alternatives to these higher-level nodes. Gene segments with higher rates of nonsynonymous change tend to be more informative overall, but those with lower rates tend to provide stronger support for deeper nodes. Higher-level nodes with bootstrap values in the 80% – 99% range for the complete data matrix are markedly more sensitive to substantial drops in their bootstrap percentages after character subsampling than those with 100% bootstrap, suggesting that these nodes are likely not to have been strongly supported with many fewer data than in the full matrix. Data set partitioning of total data by (mostly) synonymous and (mostly) nonsynonymous change improves overall node support, but the result remains much inferior to analysis of (unpartitioned) nonsynonymous change alone. Clusters of genes with similar nonsynonymous rate properties (e.g., faster vs. slower) show some distinct patterns of node support but few conflicts. Synonymous change is shown to contribute little, if any, phylogenetic signal to the support of higher-level nodes, but it does contribute nonphylogenetic signal, probably through its underlying heterogeneous nucleotide composition. Analysis of seemingly conservative indels does not prove useful.

Conclusions

Generating a robust molecular higher-level phylogeny of Arthropoda is currently possible with large amounts of data and an exclusive reliance on nonsynonymous change.     

Can deliberately incomplete gene sample augmentation improve a phylogeny estimate for the advanced moths and butterflies (Hexapoda: Lepidoptera)?

This paper addresses the question of whether one can economically improve the robustness of a molecular phylogeny estimate by increasing gene sampling in only a subset of taxa, without having the analysis invalidated by artifacts arising from large blocks of missing data. Our case study stems from an ongoing effort to resolve poorly understood deeper relationships in the large clade Ditrysia ( > 150,000 species) of the insect order Lepidoptera (butterflies and moths). Seeking to remedy the overall weak support for deeper divergences in an initial study based on five nuclear genes (6.6 kb) in 123 exemplars, we nearly tripled the total gene sample (to 26 genes, 18.4 kb) but only in a third (41) of the taxa. The resulting partially augmented data matrix (45% intentionally missing data) consistently increased bootstrap support for groupings previously identified in the five-gene (nearly) complete matrix, while introducing no contradictory groupings of the kind that missing data have been predicted to produce. Our results add to growing evidence that data sets differing substantially in gene and taxon sampling can often be safely and profitably combined. The strongest overall support for nodes above the family level came from including all nucleotide changes, while partitioning sites into sets undergoing mostly nonsynonymous versus mostly synonymous change. In contrast, support for the deepest node for which any persuasive molecular evidence has yet emerged (78-85% bootstrap) was weak or nonexistent unless synonymous change was entirely excluded, a result plausibly attributed to compositional heterogeneity. This node (Gelechioidea + Apoditrysia), tentatively proposed by previous authors on the basis of four morphological synapomorphies, is the first major subset of ditrysian superfamilies to receive strong statistical support in any phylogenetic study. A "more-genes-only" data set (41 taxa×26 genes) also gave strong signal for a second deep grouping (Macrolepidoptera) that was obscured, but not strongly contradicted, in more taxon-rich analyses.     

Comprehensive gene and taxon coverage elucidates radiation patterns in moths and butterflies

Lepidoptera (butterflies and moths) represent one of the most diverse animals groups. Yet, the phylogeny of advanced ditrysian Lepidoptera, accounting for about 99 per cent of lepidopteran species, has remained largely unresolved. We report a rigorous and comprehensive analysis of lepidopteran affinities. We performed phylogenetic analyses of 350 taxa representing nearly 90 per cent of lepidopteran families. We found Ditrysia to be a monophyletic taxon with the clade Tischerioidea + Palaephatoidea being the sister group of it. No support for the monophyly of the proposed major internested ditrysian clades, Apoditrysia, Obtectomera and Macrolepidoptera, was found as currently defined, but each of these is supported with some modification. The monophyly or near-monophyly of most previously identified lepidopteran superfamilies is reinforced, but several species-rich superfamilies were found to be para- or polyphyletic. Butterflies were found to be more closely related to ‘microlepidopteran’ groups of moths rather than the clade Macrolepidoptera, where they have traditionally been placed. There is support for the monophyly of Macrolepidoptera when butterflies and Calliduloidea are excluded. The data suggest that the generally short diverging nodes between major groupings in basal non-tineoid Ditrysia are owing to their rapid radiation, presumably in correlation with the radiation of flowering plants.     

Phylogeny of Oedogoniales, Chaetophorales and Chaetopeltidales (Chlorophyceae): inferences from sequence-structure analysis of ITS2

Background and Aims

The green algal class Chlorophyceae comprises five orders (Chlamydomonadales, Sphaeropleales, Chaetophorales, Chaetopeltidales and Oedogoniales). Attempts to resolve the relationships among these groups have met with limited success. Studies of single genes (18S rRNA, 26S rRNA, rbcL or atpB) have largely failed to unambiguously resolve the relative positions of Oedogoniales, Chaetophorales and Chaetopeltidales (the OCC taxa). In contrast, recent genomics analyses of plastid data from OCC exemplars provided a robust phylogenetic analysis that supports a monophyletic OCC alliance.

Methods

An ITS2 data set was assembled to independently test the OCC hypothesis and to evaluate the performance of these data in assessing green algal phylogeny at the ordinal or class level. Sequence-structure analysis designed for use with ITS2 data was employed for phylogenetic reconstruction.

Key Results

Results of this study yielded trees that were, in general, topologically congruent with the results from the genomic analyses, including support for the monophyly of the OCC alliance.

Conclusions

Not all nodes from the ITS2 analyses exhibited robust support, but our investigation demonstrates that sequence-structure analyses of ITS2 provide a taxon-rich means of testing phylogenetic hypotheses at high taxonomic levels. Thus, the ITS2 data, in the context of sequence-structure analysis, provide an economical supplement or alternative to the single-marker approaches used in green algal phylogeny.     

A molecular phylogeny for the leaf-roller moths (Lepidoptera: Tortricidae) and its implications for classification and life history evolution

Background

Tortricidae, one of the largest families of microlepidopterans, comprise about 10,000 described species worldwide, including important pests, biological control agents and experimental models. Understanding of tortricid phylogeny, the basis for a predictive classification, is currently provisional. We present the first detailed molecular estimate of relationships across the tribes and subfamilies of Tortricidae, assess its concordance with previous morphological evidence, and re-examine postulated evolutionary trends in host plant use and biogeography.

Methodology/Principal Findings

We sequenced up to five nuclear genes (6,633 bp) in each of 52 tortricids spanning all three subfamilies and 19 of the 22 tribes, plus up to 14 additional genes, for a total of 14,826 bp, in 29 of those taxa plus all 14 outgroup taxa. Maximum likelihood analyses yield trees that, within Tortricidae, differ little among data sets and character treatments and are nearly always strongly supported at all levels of divergence. Support for several nodes was greatly increased by the additional 14 genes sequenced in just 29 of 52 tortricids, with no evidence of phylogenetic artifacts from deliberately incomplete gene sampling. There is strong support for the monophyly of Tortricinae and of Olethreutinae, and for grouping of these to the exclusion of Chlidanotinae. Relationships among tribes are robustly resolved in Tortricinae and mostly so in Olethreutinae. Feeding habit (internal versus external) is strongly conserved on the phylogeny. Within Tortricinae, a clade characterized by eggs being deposited in large clusters, in contrast to singly or in small batches, has markedly elevated incidence of polyphagous species. The five earliest-branching tortricid lineages are all species-poor tribes with mainly southern/tropical distributions, consistent with a hypothesized Gondwanan origin for the family.

Conclusions/Significance

We present the first robustly supported phylogeny for Tortricidae, and a revised classification in which all of the sampled tribes are now monophyletic.     

Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study

Background

In the mega-diverse insect order Lepidoptera (butterflies and moths; 165,000 described species), deeper relationships are little understood within the clade Ditrysia, to which 98% of the species belong. To begin addressing this problem, we tested the ability of five protein-coding nuclear genes (6.7 kb total), and character subsets therein, to resolve relationships among 123 species representing 27 (of 33) superfamilies and 55 (of 100) families of Ditrysia under maximum likelihood analysis.

Results

Our trees show broad concordance with previous morphological hypotheses of ditrysian phylogeny, although most relationships among superfamilies are weakly supported. There are also notable surprises, such as a consistently closer relationship of Pyraloidea than of butterflies to most Macrolepidoptera. Monophyly is significantly rejected by one or more character sets for the putative clades Macrolepidoptera as currently defined (P < 0.05) and Macrolepidoptera excluding Noctuoidea and Bombycoidea sensu lato (P ≤ 0.005), and nearly so for the superfamily Drepanoidea as currently defined (P < 0.08). Superfamilies are typically recovered or nearly so, but usually without strong support. Relationships within superfamilies and families, however, are often robustly resolved. We provide some of the first strong molecular evidence on deeper splits within Pyraloidea, Tortricoidea, Geometroidea, Noctuoidea and others. Separate analyses of mostly synonymous versus non-synonymous character sets revealed notable differences (though not strong conflict), including a marked influence of compositional heterogeneity on apparent signal in the third codon position (nt3). As available model partitioning methods cannot correct for this variation, we assessed overall phylogeny resolution through separate examination of trees from each character set. Exploration of "tree space" with GARLI, using grid computing, showed that hundreds of searches are typically needed to find the best-feasible phylogeny estimate for these data.

Conclusion

Our results (a) corroborate the broad outlines of the current working phylogenetic hypothesis for Ditrysia, (b) demonstrate that some prominent features of that hypothesis, including the position of the butterflies, need revision, and (c) resolve the majority of family and subfamily relationships within superfamilies as thus far sampled. Much further gene and taxon sampling will be needed, however, to strongly resolve individual deeper nodes.     

High-throughput sequencing of six bamboo chloroplast genomes: phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae)

Background

Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction) of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies.

Methodology/Principal Findings

Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp) genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with ≥0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae.

Conclusions/Significance

The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among three clades of temperate woody bamboos, even with complete cp genome sequences, suggests that these lineages may have diverged very rapidly.     

Advances in the use of DNA barcodes to build a community phylogeny for tropical trees in a Puerto Rican forest dynamics plot

Background

Species number, functional traits, and phylogenetic history all contribute to characterizing the biological diversity in plant communities. The phylogenetic component of diversity has been particularly difficult to quantify in species-rich tropical tree assemblages. The compilation of previously published (and often incomplete) data on evolutionary relationships of species into a composite phylogeny of the taxa in a forest, through such programs as Phylomatic, has proven useful in building community phylogenies although often of limited resolution. Recently, DNA barcodes have been used to construct a robust community phylogeny for nearly 300 tree species in a forest dynamics plot in Panama using a supermatrix method. In that study sequence data from three barcode loci were used to generate a well-resolved species-level phylogeny.

Methodology/Principal Findings

Here we expand upon this earlier investigation and present results on the use of a phylogenetic constraint tree to generate a community phylogeny for a diverse, tropical forest dynamics plot in Puerto Rico. This enhanced method of phylogenetic reconstruction insures the congruence of the barcode phylogeny with broadly accepted hypotheses on the phylogeny of flowering plants (i.e., APG III) regardless of the number and taxonomic breadth of the taxa sampled. We also compare maximum parsimony versus maximum likelihood estimates of community phylogenetic relationships as well as evaluate the effectiveness of one- versus two- versus three-gene barcodes in resolving community evolutionary history.

Conclusions/Significance

As first demonstrated in the Panamanian forest dynamics plot, the results for the Puerto Rican plot illustrate that highly resolved phylogenies derived from DNA barcode sequence data combined with a constraint tree based on APG III are particularly useful in comparative analysis of phylogenetic diversity and will enhance research on the interface between community ecology and evolution.     

Comprehensive molecular sampling yields a robust phylogeny for geometrid moths (Lepidoptera: Geometridae)

Background

The moth family Geometridae (inchworms or loopers), with approximately 23 000 described species, is the second most diverse family of the Lepidoptera. Apart from a few recent attempts based on morphology and molecular studies, the phylogeny of these moths has remained largely uninvestigated.

Methodology/Principal Findings

We performed a rigorous and extensive molecular analysis of eight genes to examine the geometrid affinities in a global context, including a search for its potential sister-taxa. Our maximum likelihood analyses included 164 taxa distributed worldwide, of which 150 belong to the Geometridae. The selected taxa represent all previously recognized subfamilies and nearly 90% of recognized tribes, and originate from all over world. We found the Geometridae to be monophyletic with the Sematuridae+Epicopeiidae clade potentially being its sister-taxon. We found all previously recognized subfamilies to be monophyletic, with a few taxa misplaced, except the Oenochrominae+Desmobathrinae complex that is a polyphyletic assemblage of taxa and the Orthostixinae, which was positioned within the Ennominae. The Sterrhinae and Larentiinae were found to be sister to the remaining taxa, followed by Archiearinae, the polyphyletic assemblage of Oenochrominae+Desmobathrinae moths, Geometrinae and Ennominae.

Conclusions/Significance

Our study provides the first comprehensive phylogeny of the Geometridae in a global context. Our results generally agree with the other, more restricted studies, suggesting that the general phylogenetic patterns of the Geometridae are now well-established. Generally the subfamilies, many tribes, and assemblages of tribes were well supported but their interrelationships were often weakly supported by our data. The Eumeleini were particularly difficult to place in the current system, and several tribes were found to be para- or polyphyletic.     

Resolving Discrepancy between Nucleotides and Amino Acids in Deep-Level Arthropod Phylogenomics: Differentiating Serine Codons in 21-Amino-Acid Models

Background

In a previous study of higher-level arthropod phylogeny, analyses of nucleotide sequences from 62 protein-coding nuclear genes for 80 panarthopod species yielded significantly higher bootstrap support for selected nodes than did amino acids. This study investigates the cause of that discrepancy.

Methodology/Principal Findings

The hypothesis is tested that failure to distinguish the serine residues encoded by two disjunct clusters of codons (TCN, AGY) in amino acid analyses leads to this discrepancy. In one test, the two clusters of serine codons (Ser1, Ser2) are conceptually translated as separate amino acids. Analysis of the resulting 21-amino-acid data matrix shows striking increases in bootstrap support, in some cases matching that in nucleotide analyses. In a second approach, nucleotide and 20-amino-acid data sets are artificially altered through targeted deletions, modifications, and replacements, revealing the pivotal contributions of distinct Ser1 and Ser2 codons. We confirm that previous methods of coding nonsynonymous nucleotide change are robust and computationally efficient by introducing two new degeneracy coding methods. We demonstrate for degeneracy coding that neither compositional heterogeneity at the level of nucleotides nor codon usage bias between Ser1 and Ser2 clusters of codons (or their separately coded amino acids) is a major source of non-phylogenetic signal.

Conclusions

The incongruity in support between amino-acid and nucleotide analyses of the forementioned arthropod data set is resolved by showing that “standard” 20-amino-acid analyses yield lower node support specifically when serine provides crucial signal. Separate coding of Ser1 and Ser2 residues yields support commensurate with that found by degenerated nucleotides, without introducing phylogenetic artifacts. While exclusion of all serine data leads to reduced support for serine-sensitive nodes, these nodes are still recovered in the ML topology, indicating that the enhanced signal from Ser1 and Ser2 is not qualitatively different from that of the other amino acids.     

Pair-flowered cymes in the Lamiales: structure,distribution and origin

Background and Aims

In the Lamiales, indeterminate thyrses (made up of axillary cymes) represent a significant inflorescence type. However, it has been largely overlooked that there occur two types of cymes: (1) ordinary cymes, and (2) ‘pair-flowered cymes’ (PFCs), with a flower pair (terminal and front flower) topping each cyme unit. PFCs are unique to the Lamiales and their distribution, origin and phylogeny are not well understood.

Methods

The Lamiales are screened as to the occurrence of PFCs, ordinary cymes and single flowers (constituting racemic inflorescences).

Key Results

PFCs are shown to exhibit a considerable morphological and developmental diversity and are documented to occur in four neighbouring taxa of Lamiales: Calceolariaceae, Sanango, Gesneriaceae and Plantaginaceae. They are omnipresent in the Calceolariaceae and almost so in the Gesneriaceae. In the Plantaginaceae, PFCs are restricted to the small sister tribes Russelieae and Cheloneae (while the large remainder has single flowers in the leaf/bract axils; ordinary cymes do not occur). Regarding the origin of PFCs, the inflorescences of the genus Peltanthera (unplaced as to family; sister to Calceolariaceae, Sanango and Gesneriaceae in most molecular phylogenies) support the idea that PFCs have originated from paniculate systems, with the front-flowers representing remnant flowers.

Conclusions

From the exclusive occurrence of PFCs in the Lamiales and the proximity of the respective taxa in molecular phylogenies it may be expected that PFCs have originated once, representing a synapomorphy for this group of taxa and fading out within the Plantaginaceae. However, molecular evidence is ambiguous. Depending on the position of Peltanthera (depending in turn on the kind and number of genes and taxa analysed) a single, a double (the most probable scenario) or a triple origin appears conceivable.     

Phylogenetic analysis of pelecaniformes (aves) based on osteological data: implications for waterbird phylogeny and fossil calibration studies

Background

Debate regarding the monophyly and relationships of the avian order Pelecaniformes represents a classic example of discord between morphological and molecular estimates of phylogeny. This lack of consensus hampers interpretation of the group''s fossil record, which has major implications for understanding patterns of character evolution (e.g., the evolution of wing-propelled diving) and temporal diversification (e.g., the origins of modern families). Relationships of the Pelecaniformes were inferred through parsimony analyses of an osteological dataset encompassing 59 taxa and 464 characters. The relationships of the Plotopteridae, an extinct family of wing-propelled divers, and several other fossil pelecaniforms (Limnofregata, Prophaethon, Lithoptila, ?Borvocarbo stoeffelensis) were also assessed. The antiquity of these taxa and their purported status as stem members of extant families makes them valuable for studies of higher-level avian diversification.

Methodology/Principal Findings

Pelecaniform monophyly is not recovered, with Phaethontidae recovered as distantly related to all other pelecaniforms, which are supported as a monophyletic Steganopodes. Some anatomical partitions of the dataset possess different phylogenetic signals, and partitioned analyses reveal that these discrepancies are localized outside of Steganopodes, and primarily due to a few labile taxa. The Plotopteridae are recovered as the sister taxon to Phalacrocoracoidea, and the relationships of other fossil pelecaniforms representing key calibration points are well supported, including Limnofregata (sister taxon to Fregatidae), Prophaethon and Lithoptila (successive sister taxa to Phaethontidae), and ?Borvocarbo stoeffelensis (sister taxon to Phalacrocoracidae). These relationships are invariant when ‘backbone’ constraints based on recent avian phylogenies are imposed.

Conclusions/Significance

Relationships of extant pelecaniforms inferred from morphology are more congruent with molecular phylogenies than previously assumed, though notable conflicts remain. The phylogenetic position of the Plotopteridae implies that wing-propelled diving evolved independently in plotopterids and penguins, representing a remarkable case of convergent evolution. Despite robust support for the placement of fossil taxa representing key calibration points, the successive outgroup relationships of several “stem fossil + crown family” clades are variable and poorly supported across recent studies of avian phylogeny. Thus, the impact these fossils have on inferred patterns of temporal diversification depends heavily on the resolution of deep nodes in avian phylogeny.     

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

Background

Bivalves are very ancient and successful conchiferan mollusks (both in terms of species number and geographical distribution). Despite their importance in marine biota, their deep phylogenetic relationships were scarcely investigated from a molecular perspective, whereas much valuable work has been done on taxonomy, as well as phylogeny, of lower taxa.

Methodology/Principal Findings

Here we present a class-level bivalve phylogeny with a broad sample of 122 ingroup taxa, using four mitochondrial markers (MT-RNR1, MT-RNR2, MT-CO1, MT-CYB). Rigorous techniques have been exploited to set up the dataset, analyze phylogenetic signal, and infer a single final tree. In this study, we show the basal position of Opponobranchia to all Autobranchia, as well as of Palaeoheterodonta to the remaining Autobranchia, which we here propose to call Amarsipobranchia. Anomalodesmata were retrieved as monophyletic and basal to (Heterodonta + Pteriomorphia).

Conclusions/Significance

Bivalve morphological characters were traced onto the phylogenetic trees obtained from the molecular analysis; our analysis suggests that eulamellibranch gills and heterodont hinge are ancestral characters for all Autobranchia. This conclusion would entail a re-evaluation of bivalve symplesiomorphies.     

The fossil record and taphonomy of butterflies and moths (Insecta,Lepidoptera): implications for evolutionary diversity and divergence-time estimates

Background

It is conventionally accepted that the lepidopteran fossil record is significantly incomplete when compared to the fossil records of other, very diverse, extant insect orders. Such an assumption, however, has been based on cumulative diversity data rather than using alternative statistical approaches from actual specimen counts.

Results

We reviewed documented specimens of the lepidopteran fossil record, currently consisting of 4,593 known specimens that are comprised of 4,262 body fossils and 331 trace fossils. The temporal distribution of the lepidopteran fossil record shows significant bias towards the late Paleocene to middle Eocene time interval. Lepidopteran fossils also record major shifts in preservational style and number of represented localities at the Mesozoic stage and Cenozoic epoch level of temporal resolution. Only 985 of the total known fossil specimens (21.4%) were assigned to 23 of the 40 extant lepidopteran superfamilies. Absolute numbers and proportions of preservation types for identified fossils varied significantly across superfamilies. The secular increase of lepidopteran family-level diversity through geologic time significantly deviates from the general pattern of other hyperdiverse, ordinal-level lineages.

Conclusion

Our statistical analyses of the lepidopteran fossil record show extreme biases in preservation type, age, and taxonomic composition. We highlight the scarcity of identified lepidopteran fossils and provide a correspondence between the latest lepidopteran divergence-time estimates and relevant fossil occurrences at the superfamily level. These findings provide caution in interpreting the lepidopteran fossil record through the modeling of evolutionary diversification and in determination of divergence time estimates.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-015-0290-8) contains supplementary material, which is available to authorized users.     

A “Shallow Phylogeny” of Shallow Barnacles (Chthamalus)

Background

We present a multi-locus phylogenetic analysis of the shallow water (high intertidal) barnacle genus Chthamalus, focusing on member species in the western hemisphere. Understanding the phylogeny of this group improves interpretation of classical ecological work on competition, distributional changes associated with climate change, and the morphological evolution of complex cirripede phenotypes.

Methodology and Findings

We use traditional and Bayesian phylogenetic and ‘deep coalescent’ approaches to identify a phylogeny that supports the monophyly of the mostly American ‘fissus group’ of Chthamalus, but that also supports a need for taxonomic revision of Chthamalus and Microeuraphia. Two deep phylogeographic breaks were also found within the range of two tropical American taxa (C. angustitergum and C. southwardorum) as well.

Conclusions

Our data, which include two novel gene regions for phylogenetic analysis of cirripedes, suggest that much more evaluation of the morphological evolutionary history and taxonomy of Chthamalid barnacles is necessary. These data and associated analyses also indicate that the radiation of species in the late Pliocene and Pleistocene was very rapid, and may provide new insights toward speciation via transient allopatry or ecological barriers.     

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

Background and Aims

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids.

Methods

Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network.

Key Results

Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine.

Conclusions

Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.     

Molecular Phylogeny of the Butterfly Genus Polytremis (Hesperiidae,Hesperiinae, Baorini) in China

Background

The genus Polytremis, restricted to the continental part of the southeastern Palaearctic and northern Oriental regions, is one of the largest and most diverse lineages of the tribe Baorini. Previous studies on the genus were focused mainly on morphological classification and identification of new species. Due to the lack of effective and homologous traits of morphology, there were many challenges in the traditional classification. In this report, we reconstruct the phylogeny to provide a solid framework for these studies and to test the traditional limits and relationships of taxa.

Methodology and Principal Findings

We sequenced a mitochondrial and three nuclear gene fragments, coupled with an evaluation of traditional morphological characters, to determine the phylogenetic relationships for a total of 15 species representing all major species groups of the Polytremis genus in China, and to elucidate their taxonomic status.

Conclusions and Significance

Analysis of mitochrondial and nuclear DNA showed considerable congruent phylogenetic signal in topology at the inter-species level. We found strong support for the monophyly of Polytremis and some clades were recognized with morphological data. Thus, the COI sequence in our study could be used as a DNA barcode to identify almost all members of the genus. However, incongruences of phylogenetic analyses occurred: in contrast to the phylogenetic trees of mitochondrial COI, it was not possible for nuclear rDNA to discriminate P. gotama from P. caerulescens, suggesting a possible recent separation of these two species. Additionally, P. theca was the only species with a greater intra-specific genetic distance compared to some inter-specific genetic distances in this study and some problems associated with the cryptic diversity of the species are discussed. The results of this study will helpful to reveal the causes of the high degree of diversity of butterflies, and possibly other groups of insects in China.     

Molecular phylogeny of the neotropical genus Christensonella (Orchidaceae, Maxillariinae): species delimitation and insights into chromosome evolution

Background and Aims

Species'' boundaries applied within Christensonella have varied due to the continuous pattern of variation and mosaic distribution of diagnostic characters. The main goals of this study were to revise the species'' delimitation and propose a more stable classification for this genus. In order to achieve these aims phylogenetic relationships were inferred using DNA sequence data and cytological diversity within Christensonella was examined based on chromosome counts and heterochromatin patterns. The results presented describe sets of diagnostic morphological characters that can be used for species'' identification.

Methods

Phylogenetic studies were based on sequence data of nuclear and plastid regions, analysed using maximum parsimony and maximum likelihood criteria. Cytogenetic observations of mitotic cells were conducted using CMA and DAPI fluorochromes.

Key Results

Six of 21 currently accepted species were recovered. The results also support recognition of the ‘C. pumila’ clade as a single species. Molecular phylogenetic relationships within the ‘C. acicularis–C. madida’ and ‘C. ferdinandiana–C. neowiedii’ species'' complexes were not resolved and require further study. Deeper relationships were incongruent between plastid and nuclear trees, but with no strong bootstrap support for either, except for the position of C. vernicosa. Cytogenetic data indicated chromosome numbers of 2n = 36, 38 and 76, and with substantial variation in the presence and location of CMA/DAPI heterochromatin bands.

Conclusions

The recognition of ten species of Christensonella is proposed according to the molecular and cytogenetic patterns observed. In addition, diagnostic morphological characters are presented for each recognized species. Banding patterns and chromosome counts suggest the occurrence of centric fusion/fission events, especially for C. ferdinandiana. The results suggest that 2n = 36 karyotypes evolved from 2n = 38 through descendent dysploidy. Patterns of heterochromatin distribution and other karyotypic data proved to be a valuable source of information to understand evolutionary patterns within Maxillariinae orchids.Key words: Chromosome number, Christensonella, Cymbidieae, cytotaxonomy, fluorochrome staining, Maxillaria, Maxillariinae, molecular phylogenetics, species delimitation     

Combining FISH and model-based predictions to understand chromosome evolution in Typhonium (Araceae)

Background and Aims

Since the advent of molecular phylogenetics, numerous attempts have been made to infer the evolutionary trajectories of chromosome numbers on DNA phylogenies. Ideally, such inferences should be evaluated against cytogenetic data. Towards this goal, we carried out phylogenetic modelling of chromosome number change and fluorescence in situ hybridization (FISH) in a medium sized genus of Araceae to elucidate if data from chromosomal markers would support maximum likelihood-inferred changes in chromosome numbers among close relatives. Typhonium, the focal genus, includes species with 2n = 65 and 2n = 8, the lowest known count in the family.

Methods

A phylogeny from nuclear and plastid sequences (96 taxa, 4252 nucleotides) and counts for all included species (15 of them first reported here) were used to model chromosome number evolution, assuming discrete events, such as polyploidization and descending or ascending dysploidy, occurring at different rates. FISH with three probes (5S rDNA, 45S rDNA and Arabidopsis-like telomeres) was performed on ten species with 2n = 8 to 2n = 24.

Key Results

The best-fitting models assume numerous past chromosome number reductions. Of the species analysed with FISH, the two with the lowest chromosome numbers contained interstitial telomeric signals (Its), which together with the phylogeny and modelling indicates decreasing dysploidy as an explanation for the low numbers. A model-inferred polyploidization in another species is matched by an increase in rDNA sites.

Conclusions

The combination of a densely sampled phylogeny, ancestral state modelling and FISH revealed that the species with n = 4 is highly derived, with the FISH data pointing to a Robertsonian fusion-like chromosome rearrangement in the ancestor of this species.