Application of multigene phylogenetics and site‐stripping to resolve intraordinal relationships in the Rhodymeniales (Rhodophyta)

Previous molecular assessments of the red algal order Rhodymeniales have confirmed its monophyly and distinguished the six currently recognized families (viz. Champiaceae, Faucheaceae, Fryeellaceae, Hymenocladiaceae, Lomentariaceae, and Rhodymeniaceae); however, relationships among most of these families have remained unresolved possibly as a result of substitution saturation at deeper phylogenetic nodes. The objective of the current study was to improve rhodymenialean systematics by increasing taxonomic representation and using a more robust multigene dataset of mitochondrial (COB, COI/COI‐5P), nuclear (LSU, EF2) and plastid markers (psbA, rbcL). Additionally, we aimed to prevent phylogenetic inference problems associated with substitution saturation (particularly at the interfamilial nodes) by removing fast‐evolving sites and analyzing a series of progressively more conservative alignments. The Rhodymeniales was resolved as two major lineages: (i) the Fryeellaceae as sister to the Faucheaceae and Lomentariaceae; and (ii) the Rhodymeniaceae allied to the Champiaceae and Hymenocladiaceae. Support at the interfamilial nodes was highest when 20% of variable sites were removed. Inclusion of Binghamiopsis, Chamaebotrys, and Minium, which were absent in previous phylogenetic investigations, established their phylogenetic affinities while assessment of two genera consistently polyphyletic in phylogenetic analyses, Erythrymenia and Lomentaria, resulted in the proposition of the novel genera Perbella and Fushitsunagia. The taxonomic position of Drouetia was reinvestigated with re‐examination of holotype material of D. coalescens to clarify tetrasporangial development in this genus. In addition, we added three novel Australian species to Drouetia as a result of ongoing DNA barcoding assessments—D. aggregata sp. nov., D. scutellata sp. nov., and D. viridescens sp. nov.     

Revisiting Ross and Sims (1971): toward a molecular phylogeny of the Biddulphiaceae and Eupodiscaceae (Bacillariophyceae)

The ocellate and pseudocellate diatoms in the Eupodiscaceae and Biddulphiaceae (respectively) are common inhabitants of the marine littoral (and plankton zone) with a rich fossil history making them important components of marine stratigraphic studies and good candidates for molecular dating work. These diatoms are important for un‐derstanding the phylogeny of the diatoms as a whole, as molecular phylogenies have blurred the traditional distinction between the pennate and multipolar non‐pennate diatoms. However, the convoluted taxonomic history of these groups has the potential to disrupt both stratigraphic and molecular dating studies. Although efforts have been made to examine frustule morphology of several ocellate and pseudocellate diatoms and develop a morphological scheme to define genera, very little work has been done to determine how these groups are interrelated. In this study, we use nuclear and chloroplast molecular markers to construct a phylogeny of a diverse sampling of Eupodiscaceae and Biddulphiaceae taxa. The ocellus‐bearing taxa (Eupodiscaceae) are monophyletic, and thus the ocellus may be a useful character in delimiting the Eupodiscaceae, the Biddulphiaceae are polyphyletic and scattered across a number of lineages of multipolar non‐pennate diatoms. Hypothesis testing aimed at assessing the likeliness of several morphology based hypotheses against the molecular data highlights uncertainty in both types of data. We present evidence that there are monophyletic genera within both the Biddulphiaceae and Eupodiscaceae, and recommend the taxa within the Odontella mobilensis/sinensis/regia clade be transferred to a new genus: Trieres Ashworth & Theriot.     

The evolutionary history of Afrocanarian blue tits inferred from genomewide SNPs

A common challenge in phylogenetic reconstruction is to find enough suitable genomic markers to reliably trace splitting events with short internodes. Here, we present phylogenetic analyses based on genomewide single‐nucleotide polymorphisms (SNPs) of an enigmatic avian radiation, the subspecies complex of Afrocanarian blue tits (Cyanistes teneriffae). The two sister species, the Eurasian blue tit (Cyanistes caeruleus) and the azure tit (Cyanistes cyanus), constituted the out‐group. We generated a large data set of SNPs for analysis of population structure and phylogeny. We also adapted our protocol to utilize degraded DNA from old museum skins from Libya. We found strong population structuring that largely confirmed subspecies monophyly and constructed a coalescent‐based phylogeny with full support at all major nodes. The results are consistent with a recent hypothesis that La Palma and Libya are relic populations of an ancient Afrocanarian blue tit, although a small data set for Libya could not resolve its position relative to La Palma. The birds on the eastern islands of Fuerteventura and Lanzarote are similar to those in Morocco. Together they constitute the sister group to the clade containing the other Canary Islands (except La Palma), in which El Hierro is sister to the three central islands. Hence, extant Canary Islands populations seem to originate from multiple independent colonization events. We also found population divergences in a key reproductive trait, viz. sperm length, which may constitute reproductive barriers between certain populations. We recommend a taxonomic revision of this polytypic species, where several subspecies should qualify for species rank.     

Diversity of teiid lizards from Gran Chaco and Western Cerrado (Squamata: Teiidae)

The Gran Chaco dry forest ecoregion corresponds to the southern portion of the South America diagonal belt of open formations, being one of the most threatened subtropical woodland savannas in the world. The area is still poorly known biologically and has been suffering with impressively high forest cover loss in the last 10 years. Integrating morphological and molecular data, we detected and describe a cryptic new species of lizard genus Ameivula endemic from the eastern part of this ecoregion, the called Humid Chaco. Ameivula apipensis sp nov. is characterised by a whitish brown vertebral stripe in adults and juveniles, a lateral field without ocelli and with overlapping spot, presence of an interfrontoparietal scale in 46.2% of the specimens, 12–17 femoral pores, an hemipenis without lateral sac, five xiphisternal ribs, and by a combination of meristic features as confirmed by discriminant analysis. The new species was recovered sister to a clade from Western Cerrado in our analysis, the first phylogenetic hypothesis for the Ameivula and Glaucomastix genera based on 1977 base pairs of three mitochondrial (12S, 16S and cyt‐b) and one nuclear (c‐mos) genes, including all the recognised species at the moment. Maximum parsimony and Bayesian inference recovered the monophyly of Ameivula and Glaucomastix with strong support. Reinforcing previous studies, our results suggest the presence of additional cryptic species in Ameivula from the Western Cerrado.     

Sensitive phylogenetics of Clematis and its position in Ranunculaceae

Ranunculaceae are a nearly cosmopolitan plant family with the highest diversity in northern temperate regions and with relatively few representatives in the tropics. As a result of their position among the early diverging eudicots and their horticultural value, the family is of great phylogenetic and taxonomic interest. Despite this, many genera remain poorly sampled in phylogenetic studies and taxonomic problems persist. In this study, we aim to clarify the infrageneric relationships of Clematis by greatly improving taxon sampling and including most of the relevant subgeneric and sectional types in a simultaneous dynamic optimization of phenotypic and molecular data. We also investigate how well the available data support the hypothesis of phylogenetic relationships in the family. At the family level, all five currently accepted subfamilies are resolved as monophyletic. Our analyses strongly imply that Anemone s.l. is a grade with respect to the Anemoclema + Clematis clade. This questions the recent sinking of well‐established genera, including Hepatica, Knowltonia and Pulsatilla, into Anemone. In Clematis, 12 clades conceptually matching the proposed sectional division of the genus were found. The taxonomic composition of these clades often disagrees with previous classifications. Phylogenetic relationships between the section‐level clades remain highly unstable and poorly supported and, although some patterns are emerging, none of the proposed subgenera is in evidence. The traditionally recognized and horticulturally significant section Viorna is both nomenclaturally invalid and phylogenetically unsupported. Several other commonly used sections are likewise unjustified. Our results provide a phylogenetic background for a natural section‐level classification of Clematis.     

Evolution,biogeography and systematics of the western Palaearctic Zamenis ratsnakes

The phylogenetic relationships between western Palaearctic Zamenis and Rhinechis ratsnakes have been troubled, with recent estimates based on the supermatrix approach questioning their monophyly and providing contradictory results. In this study, we generated a comprehensive molecular data set for Zamenis and closely related ratsnakes to assess their phylogenetic and systematic relationships and infer their spatial and temporal modes of diversification. We obtained a fully resolved and well‐supported phylogeny, which is consistent across markers, taxon‐sets and phylogenetic methods. The close phylogenetic relationship between Rhinechis and Zamenis is well‐established. However, the early branching pattern within this clade, and the position of R. scalaris and Z. hohenackeri, remains poorly supported. The Persian ratsnake Z. persicus is sister to the Mediterranean species Z. situla, Z. longissimus and Z. lineatus, of which Z. situla is sister to a clade containing the latter two species. These results are consistent with a recent phylogenomic study on ratsnakes based on hundreds of loci. Whereas, topological tests based on our data and evidence from such phylogenomic study strongly rejected previous phylogenetic estimates based on the supermatrix approach and demonstrate that these “mega‐phylogenies”, with hundreds of taxa and high levels of missing data, have recovered inconsistent relationships with spurious nodal support. Biogeographical and molecular dating analyses suggest an origin of the ancestor of Rhinechis and Zamenis in the Aegean region with early cladogenesis during the Late Miocene associated with the Aegean arch formation and support a scenario of east‐to‐west diversification. Finally, while we have little morphological and phylogenetic evidence for the distinctiveness between Rhinechis and Zamenis, a classification of them in a single genus, and the designation of Zamenis scalaris (Schinz, 1822), reflects better their evolutionary relationships.     

Phylogenetic relationships within Adiaphanida (phylum Platyhelminthes) and the status of the crustacean‐parasitic genus Genostoma

The existence of the platyhelminth clade Adiaphanida—an assemblage comprising the well‐studied order Tricladida as well as two lesser known taxa, Prolecithophora and the obligate parasitic Fecampiida—is among the more surprising results of flatworm molecular systematics. Each of these three clades is itself largely well‐defined from a morphological point of view, although Adiaphanida at large, despite its strong support in molecular phylogenetic analyses, lacks known morphological synapomorphies. However, one taxon, the genus Genostoma, a parasite of the leptostracan crustacean Nebalia, rests uneasily within its current classification within the fecampiid family Genostomatidae; ultrastructural investigations on this taxon have uncovered a spermatogenesis reminiscent of Kalyptorhynchia, and a dorsal syncytium resembling the neodermatan tegument. Here, we provide molecular sequence data (nearly complete 18S and 28S rRNA) from a representative of Genostoma, with which we test hypotheses on the phylogenetic position of this taxon within Platyhelminthes, expanding upon a recently published phylum‐wide analysis, and applying novel alignment algorithms and substitution models. These analyses unequivocally position Genostoma as the sister group of Prolecithophora. However, even in taxon‐rich analyses, support for the position of the root of Adiaphanida is lacking, highlighting the need for new data types to study the phylogeny of this clade. Interestingly, our analyses also do not recover the monophyly of several taxa previously proposed, notably Continenticola within Tricladida and Protomonotresidae within Prolecithophora. In light of this phylogeny and the distinctive morphology (especially, spermatogenesis) of Genostoma, we advocate for a redefinition of the family Genostomatidae, outside of both Fecampiida and Prolecithophora, to encompass the members of this unique genus of parasites. Within Fecampiida, the family Piscinquilinidae fam. nov. is erected to accommodate the vertebrate‐parasitic Piscinquilinus, formerly Genostomatidae.     

The use of DNA barcodes to estimate phylogenetic diversity in forest communities of southern China

To elucidate potential ecological and evolutionary processes associated with the assembly of plant communities, there is now widespread use of estimates of phylogenetic diversity that are based on a variety of DNA barcode regions and phylogenetic construction methods. However, relatively few studies consider how estimates of phylogenetic diversity may be influenced by single DNA barcodes incorporated into a sequence matrix (conservative regions vs. hypervariable regions) and the use of a backbone family‐level phylogeny. Here, we use general linear mixed‐effects models to examine the influence of different combinations of core DNA barcodes (rbcL, matK, ITS, and ITS2) and phylogeny construction methods on a series of estimates of community phylogenetic diversity for two subtropical forest plots in Guangdong, southern China. We ask: (a) What are the relative influences of single DNA barcodes on estimates phylogenetic diversity metrics? and (b) What is the effect of using a backbone family‐level phylogeny to estimate topology‐based phylogenetic diversity metrics? The combination of more than one barcode (i.e., rbcL + matK + ITS) and the use of a backbone family‐level phylogeny provided the most parsimonious explanation of variation in estimates of phylogenetic diversity. The use of a backbone family‐level phylogeny showed a stronger effect on phylogenetic diversity metrics that are based on tree topology compared to those that are based on branch lengths. In addition, the variation in the estimates of phylogenetic diversity that was explained by the top‐rank models ranged from 0.1% to 31% and was dependent on the type of phylogenetic community structure metric. Our study underscores the importance of incorporating a multilocus DNA barcode and the use of a backbone family‐level phylogeny to infer phylogenetic diversity, where the type of DNA barcode employed and the phylogenetic construction method used can serve as a significant source of variation in estimates of phylogenetic community structure.     

Molecular Phylogeny of Mobilid and Sessilid Ciliates Symbiotic in Eastern Pacific Limpets (Mollusca: Patellogastropoda)

The phylogenetic relationships of the ciliate subclass Peritrichia, composed of the orders Mobilida and Sessilida, have recently come under debate as morphological and molecular analyses have struck contrasting conclusions as to the monophyly of the group. We provide additional molecular data to assess the monophyly of the Peritrichia by sequencing the small subunit ribosomal RNA genes of two symbiotic peritrichs, Urceolaria korschelti and Scyphidia ubiquita, found inhabiting the mantle cavity of limpets. Although phylogenetic analyses indicated a nonmonophyletic Peritrichia, approximately unbiased tests revealed that the monophyletic hypothesis could not be rejected. With regard to the Mobilida, our analysis showed divergence within the family Trichodinidae related to host taxa—a molluscan clade and a fish clade. For the Sessilida, the family Scyphidiidae was sister to the Astylozoidae. In our sampling of U. korschelti and S. ubiquita, both species showed significant genetic divergence among geographically isolated, yet morphologically indistinguishable populations. We hypothesize that cryptic speciation has produced these morphologically identical species and argue that more extensive genomic analyses are required to fully assess the monophyly, biogeography, and ultimately biodiversity of the peritrichs.     

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.     

Revision and phylogenetic assessment of the rove beetle genus Pseudohesperus Hayashi,with broad reference to the subtribe Philonthina (Coleoptera: Staphylinidae: Staphylinini)

This paper studies the phylogeny of the rove beetle subtribe Philonthina, to test its hypothetical monophyly and to unravel the evolutionary relationships of the subtribe and its included genus‐level taxa, with emphasis on the genus Pseudohesperus and its close‐allied relatives. The phylogenetic analyses are based on 105 adult morphological characters and 66 terminal taxa, i.e., all six members of Pseudohesperus, 51 species to represent 29 other genera of the subtribe Philonthina, seven species to represent the other six subtribes of Staphylinini, one species of the tribes Arrowinini, and one of the Platyprosopini. According to the phylogenetic results obtained, the genus Erichsonius should move out from the hitherto‐defined subtribe Philonthina and thus the monophyly of this taxon is challenged. The phylogenetic tree suggests that the genera Hesperus and Belonuchus might not be monophyletic, but the monophyly of Pseudohesperus and the sister relationship between it and Bisnius are well supported. The species‐level phylogenetic relationships of the genus Pseudohesperus reveal a clear pattern of species diversification that can be correlated well with the species' zoogeographical patterns. The paper also revises the taxonomy of Pseudohesperus and describes five new species from China: Pseudohesperus luteus Li & Zhou sp. nov. , Pseudohesperus pedatiformis Li & Zhou sp. nov. , Pseudohesperus tripartitus Li & Zhou sp. nov. , Pseudohesperus sparsipunctatus Li & Zhou sp. nov. , and Bisnius lubricus Li & Zhou sp. nov. An identification key to the species of Pseudohesperus is provided and their geographical distributions are mapped. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 679–722.     

Four New Members of Foliicolous Green Algae Within the Watanabea Clade (Trebouxiophyceae,Chlorophyta) from China

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores’ morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores.     

Phylogenetics and comparative analysis of the mitochondrial genomes of three violet‐ringed octopuses

Similar morphological characters and little molecular data of Amphioctopus rex, A. neglectus and A. cf. ovulum resulted in their unknown phylogenetic statuses and equivocal relationships. In this study, the complete mitochondrial genomes of these three species collected in Chinese waters were sequenced and compared with each other to clarify the relationships among them. The lengths of the mitochondrial genomes varied from 15,646 bp to 15,814 bp, and the A + T content and GC skew for protein‐coding genes showed little variation. In contrast, both a dendrogram based on codon usage and the gene arrangements of the three octopuses showed that A. rex was more closely related to A. neglectus than to A. cf. ovulum. Five data sets and two methods (maximum likelihood and Bayesian inference) were utilized for the first time to explore the phylogenetic relationships among these three species in Octopodidae. The results indicated that a data set combining protein‐coding genes and RNA genes (PR) was optimal for analysing the relationships among 43 cephalopods. All of the phylogenetic trees divided the cephalopods into 10 taxa and supported the monophyly of Oegopsida, Myopsida, Sepiidae and Octopodidae. In this study, Idiosepiidae was classified as sister to Sepiolidae. Trees constructed using all data sets robustly supported the monophyly of the genus Amphioctopus. Notably, A. rex was more closely related to A. neglectus than to A. cf. ovulum, although these three species share the characteristic of violet rings on dark ocelli.     

A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish, and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings and terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM, and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear‐encoded SSU (18S) rDNA and chloroplast‐encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under‐scoring of this genus in marine plankton.     

Interspecific plastidial recombination in the diatom genus Pseudo‐nitzschia

Plastids are usually uni‐parentally inherited and genetic recombination between these organelles is seldom observed. The genus Pseudo‐nitzschia, a globally relevant marine diatom, features bi‐parental plastid inheritance in the course of sexual reproduction. This observation inspired the recombination detection we pursued in this paper over a ~1,400‐nucleotide‐long region of the plastidial rbcL, a marker used in both molecular taxonomy and phylogenetic studies in diatoms. Among all the rbcL‐sequences available in web‐databases for Pseudo‐nitzschia, 42 haplotypes were identified and grouped in five clusters by Bayesian phylogeny. Signs of hybridization were evident in four of five clusters, at both intra‐ and interspecific levels, suggesting that, in diatoms, (i) plastidial recombination is not absent and (ii) hybridization can play a role in speciation of Pseudo‐nitzschia spp.