Intragenomic variation of the rDNA internal transcribed spacers in sponges (Phylum Porifera): implications for phylogenetic studies

The internal transcribed spacer regions (ITS1 and ITS2) of the tandemly repeated nuclear ribosomal DNA clusters are frequently used as markers for fine scale analyses in diverse animals. In certain taxa, ITS is nearly exclusively used for population level or inter-specific studies, despite the frequent presence of divergent paralogs within individual genomes that can be phylogenetically misleading. For the first time we survey diverse marine sponges to determine the extent and phylogenetic implications of intragenomic polymorphisms (IGPs) exhibited at their ITS loci. We discover that the extent of IGP varies greatly between taxa (with most taxa exhibiting very few) and cannot be predicted by taxonomy. Furthermore, we demonstrate that ITS can be phylogenetically informative between species when moderate levels of IGPs are detected, but that ITS paralogy can interfere with population level studies. We caution against the routine use of ITS in phylogenetic studies of sponges without (1) screening for IGPs in specimens from every population sampled; (2) including all divergent paralogs in phylogenetic analyses; (3) testing ITS data using other single-copy, unlinked loci (such as nuclear introns).     

First reported occurrence of wewokellid sponges (Calcarea, Heteractinida) from the Permian of central Iran

The new calcisponges Regispongia fluegeli n. sp., and Iranospongia circulara n. gen. n. sp., are described from central Iran. These are the first heteractinid sponges reported from the Permian of the region. These wewokellid sponges are large, irregularly cylindrical forms with a distinct axial spongocoel. The calcareous spicular skeletons of both taxa have been overgrown and are recrystallized. However, the preserved skeleton of Regispongia fluegeli does include large polyactines in the main endosomal layer and small octactines and possibly other polyactine spicules in both the relatively massive dermal layer and the distinct, delicately spiculed, gastral layer. Iranospongia is characterized by a discontinuous ring of vertical exhalant canals interior to the dense dermal layer, and by an interior skeleton net that includes common coarse vertical fibers. Individual spicules in Iranospongia are commonly obscured, but locally some remnants of possible polyactines occur in outer parts of the skeleton.     

Molecular phylogenetic evaluation of classification and scenarios of character evolution in calcareous sponges (Porifera, Class Calcarea)

Calcareous sponges (Phylum Porifera, Class Calcarea) are known to be taxonomically difficult. Previous molecular studies have revealed many discrepancies between classically recognized taxa and the observed relationships at the order, family and genus levels; these inconsistencies question underlying hypotheses regarding the evolution of certain morphological characters. Therefore, we extended the available taxa and character set by sequencing the complete small subunit (SSU) rDNA and the almost complete large subunit (LSU) rDNA of additional key species and complemented this dataset by substantially increasing the length of available LSU sequences. Phylogenetic analyses provided new hypotheses about the relationships of Calcarea and about the evolution of certain morphological characters. We tested our phylogeny against competing phylogenetic hypotheses presented by previous classification systems. Our data reject the current order-level classification by again finding non-monophyletic Leucosolenida, Clathrinida and Murrayonida. In the subclass Calcinea, we recovered a clade that includes all species with a cortex, which is largely consistent with the previously proposed order Leucettida. Other orders that had been rejected in the current system were not found, but could not be rejected in our tests either. We found several additional families and genera polyphyletic: the families Leucascidae and Leucaltidae and the genus Leucetta in Calcinea, and in Calcaronea the family Amphoriscidae and the genus Ute. Our phylogeny also provided support for the vaguely suspected close relationship of several members of Grantiidae with giantortical diactines to members of Heteropiidae. Similarly, our analyses revealed several unexpected affinities, such as a sister group relationship between Leucettusa (Leucaltidae) and Leucettidae and between Leucascandra (Jenkinidae) and Sycon carteri (Sycettidae). According to our results, the taxonomy of Calcarea is in desperate need of a thorough revision, which cannot be achieved by considering morphology alone or relying on a taxon sampling based on the current classification below the subclass level.     

Accounting for variation of substitution rates through time in Bayesian phylogeny reconstruction of Sapotoideae (Sapotaceae)

We used Bayesian phylogenetic analysis of 5 kb of chloroplast DNA data from 68 Sapotaceae species to clarify phylogenetic relationships within Sapotoideae, one of the two major clades within Sapotaceae. Variation in substitution rates through time was shown to be a very important aspect of molecular evolution for this data set. Relative rates tests indicated that changes in overall rate have taken place in several lineages during the history of the group and Bayes factors strongly supported a covarion model, which allows the rate of a site to vary over time, over commonly used models that only allow rates to vary across sites. Rate variation over time was actually found to be a more important model component than rate variation across sites. The covarion model was originally developed for coding gene sequences and has so far only been tested for this type of data. The fact that it performed so well with the present data set, consisting mainly of data from noncoding spacer regions, suggests that it deserves a wider consideration in model based phylogenetic inference. Repeatability of phylogenetic results was very difficult to obtain with the more parameter rich models, and analyses with identical settings often supported different topologies. Overparameterization may be the reason why the MCMC did not sample from the posterior distribution in these cases. The problem could, however, be overcome by using less parameter rich evolutionary models, and adjusting the MCMC settings. The phylogenetic results showed that two taxa, previously thought to belong in Sapotoideae, are not part of this group. Eberhardtia aurata is the sister of the two major Sapotaceae clades, Chrysophylloideae and Sapotoideae, and Neohemsleya usambarensis belongs in Chrysophylloideae. Within Sapotoideae two clades, Sideroxyleae and Sapoteae, were strongly supported. Bayesian analysis of the character history of some floral morphological traits showed that the ancestral type of flower in Sapotoideae may have been characterized by floral parts (sepals, petals, stamens, and staminodes) in single whorls of five, entire corolla lobes, and seeds with an adaxial hilum.     

Phylogenetic analysis of anostracans (Branchiopoda: Anostraca) inferred from nuclear 18S ribosomal DNA (18S rDNA) sequences

The nuclear small subunit ribosomal DNA (18S rDNA) of 27 anostracans (Branchiopoda: Anostraca) belonging to 14 genera and eight out of nine traditionally recognized families has been sequenced and used for phylogenetic analysis. The 18S rDNA phylogeny shows that the anostracans are monophyletic. The taxa under examination form two clades of subordinal level and eight clades of family level. Two families the Polyartemiidae and Linderiellidae are suppressed and merged with the Chirocephalidae, of which together they form a subfamily. In contrast, the Parartemiinae are removed from the Branchipodidae, raised to family level (Parartemiidae) and cluster as a sister group to the Artemiidae in a clade defined here as the Artemiina (new suborder). A number of morphological traits support this new suborder. The Branchipodidae are separated into two families, the Branchipodidae and Tanymastigidae (new family). The relationship between Dendrocephalus and Thamnocephalus requires further study and needs the addition of Branchinella sequences to decide whether the Thamnocephalidae are monophyletic. Surprisingly, Polyartemiella hazeni and Polyartemia forcipata ("Family" Polyartemiidae), with 17 and 19 thoracic segments and pairs of trunk limb as opposed to all other anostracans with only 11 pairs, do not cluster but are separated by Linderiella santarosae ("Family" Linderiellidae), which has 11 pairs of trunk limbs. All appear to be part of the Chirocephalidae and share one morphological character: double pre-epipodites on at least part of their legs. That Linderiella is part of the Polyartemiinae suggests that multiplication of the number of limbs occurred once, but was lost again in Linderiella. Within Chirocephalidae, we found two further clades, the Eubranchipus-Pristicephalus clade and the Chirocephalus clade. Pristicephalus is reinstated as a genus.     

Increased taxon sampling provides new insights into the phylogeny and evolution of the subclass Calcaronea (Porifera,Calcarea)

Calcaronean sponges are acknowledged to be taxonomically difficult, and generally, molecular data does not support the current morphology-based classification. In addition, molecular markers that have been successfully employed in other sponge taxa (e.g., COI mtDNA) have proven challenging to amplify due to the characteristics of calcarean mitochondrial genomes. A short fragment of the 28S rRNA gene (C-region) was recently proposed as the most phylogenetically informative marker to be used as a DNA barcode for calcareous sponges. In this study, the C-region and a fragment of the 18S rRNA gene were sequenced for a wide range of calcareous taxa, mainly from the subclass Calcaronea. The resulting dataset includes the most comprehensive taxon sampling of Calcaronea to date, and the inclusion of multiple specimens per species allowed us to evaluate the performance of both markers, as barcoding markers. 18S proved to be highly conserved within Calcaronea and does not have sufficient signal to resolve phylogenetic relationships within the subclass. Although the C-region does not exhibit a “proper” barcoding gap, it provides good phylogenetic resolution for calcaronean sponges. The resulting phylogeny supports previous findings that the current classification of the subclass Calcaronea is highly artificial, and with high levels of homoplasy. Furthermore, the close relationship between the order Baerida and the genus Achramorpha suggest that the order Baerida should be abandoned. Although the C-region currently represents the best available marker for phylogenetic and barcoding studies in Calcaronea, it is necessary to develop additional molecular markers to improve the classification within this subclass.     

Molecular phylogeny of glass sponges (Porifera, Hexactinellida): increased taxon sampling and inclusion of the mitochondrial protein-coding gene, cytochrome oxidase subunit I

Marine sponges of the class Hexactinellida (glass sponges) are among the most understudied groups of Porifera, and molecular approaches to investigating their evolution have only recently emerged. Although these first results appeared reliable as they largely corroborated morphology-based hypotheses, they were almost exclusively based on ribosomal RNA genes (rDNA) and should, therefore, be further tested with independent types of genetic data, such as protein-coding genes. To this end, we established the mitochondrial-encoded cytochrome oxidase subunit I gene (COI) as an additional marker, and conducted phylogenetic analyses on DNA- and amino-acid level, as well as a supermatrix analysis based on combined COI DNA and rDNA alignments. Furthermore, we increased taxon sampling compared to previous studies by adding seven additional species. The COI-based phylogenies were largely congruent with the rDNA-based phylogeny but suffered from poor bootstrap support for many nodes. However, addition of the COI sequences to the rDNA data set increased resolution of the overall molecular phylogeny. Thus, although obtaining COI sequences from glass sponges turned out to be quite challenging, this gene appears to be a valuable supplement to rDNA data for molecular evolutionary studies of this group. Some implications of our extended phylogeny for the evolution and systematics of Hexactinellida are discussed.     

A phylogeny of the genus Nocardia deduced from the analysis of small-subunit ribosomal DNA sequences, including transfer of Nocardia amarae to the genus Gordona as Gordona amarae comb. nov.

Abstract According to phylogenetic analyses of nearly complete small-subunit ribosomal DNA sequences, the genus Nocardia should not comprise the two species Nocardia petroleophila and Nocardia amarae. N. amarae should be reassigned to the genus Gordona as Gordona amarae . All of the other Nocardia species form a monophyletic unit, closely related to species of the genus Rhodococcus . It is proposed to revive the name 'CMN' to comprise the genera Corynebacterium, Tsukamurella, Mycobacterium, Gordona, Rhodococcus and Nocardia that form a well identified and monophyletic unit. They are all characterized by a cell wall chemotype IV with mycolic acids.     

Phylogeography of western Pacific Leucetta 'chagosensis' (Porifera: Calcarea) from ribosomal DNA sequences: implications for population history and conservation of the Great Barrier Reef World Heritage Area (Australia)

Leucetta 'chagosensis' is a widespread calcareous sponge, occurring in shaded habitats of Indo-Pacific coral reefs. In this study we explore relationships among 19 ribosomal DNA sequence types (the ITS1-5.8S-ITS2 region plus flanking gene sequences) found among 54 individuals from 28 locations throughout the western Pacific, with focus on the Great Barrier Reef (GBR). Maximum parsimony analysis revealed phylogeographical structuring into four major clades (although not highly supported by bootstrap analysis) corresponding to the northern/central GBR with Guam and Taiwan, the southern GBR and subtropical regions south to Brisbane, Vanuatu and Indonesia. Subsequent nested clade analysis (NCA) confirmed this structure with a probability of > 95%. After NCA of geographical distances, a pattern of range expansion from the internal Indonesian clade was inferred at the total cladogram level, as the Indonesian clade was found to be the internal and therefore oldest clade. Two distinct clades were found on the GBR, which narrowly overlap geographically in a line approximately from the Whitsunday Islands to the northern Swain Reefs. At various clade levels, NCA inferred that the northern GBR clade was influenced by past fragmentation and contiguous range expansion events, presumably during/after sea level low stands in the Pleistocene, after which the northern GBR might have been recolonized from the Queensland Plateau in the Coral Sea. The southern GBR clade is most closely related to subtropical L. 'chagosensis', and we infer that the southern GBR probably was recolonized from there after sea level low stands, based on our NCA results and supported by oceanographic data. Our results have important implications for conservation and management of the GBR, as they highlight the importance of marginal transition zones in the generation and maintenance of species rich zones, such as the Great Barrier Reef World Heritage Area.     

Performance of four ribosomal DNA regions to infer higher-level phylogenetic relationships of inoperculate euascomycetes (Leotiomyceta)

The inoperculate euascomycetes are filamentous fungi that form saprobic, parasitic, and symbiotic associations with a wide variety of animals, plants, cyanobacteria, and other fungi. The higher-level relationships of this economically important group have been unsettled for over 100 years. A data set of 55 species was assembled including sequence data from nuclear and mitochondrial small and large subunit rDNAs for each taxon; 83 new sequences were obtained for this study. Parsimony and Bayesian analyses were performed using the four-region data set and all 14 possible subpartitions of the data. The mitochondrial LSU rDNA was used for the first time in a higher-level phylogenetic study of ascomycetes and its use in concatenated analyses is supported. The classes that were recognized in Leotiomyceta (=inoperculate euascomycetes) in a classification by Eriksson and Winka [Myconet 1 (1997) 1] are strongly supported as monophyletic. The following classes formed strongly supported sister-groups: Arthoniomycetes and Dothideomycetes, Chaetothyriomycetes and Eurotiomycetes, and Leotiomycetes and Sordariomycetes. Nevertheless, the backbone of the euascomycete phylogeny remains poorly resolved. Bayesian posterior probabilities were always higher than maximum parsimony bootstrap values, but converged with an increase in gene partitions analyzed in concatenated analyses. Comparison of five recent higher-level phylogenetic studies in ascomycetes demonstrates a high degree of uncertainty in the relationships between classes.     

Low genetic structuring among <Emphasis Type="Italic">Pericharax heteroraphis</Emphasis> (Porifera: Calcarea) populations from the Great Barrier Reef (Australia), revealed by analysis of nrDNA and nuclear intron sequences

A new nuclear marker system for sponges, the second intron of the nuclear ATP synthetase beta subunit gene (ATPSbeta-iII), was analysed together with nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences aiming to uncover phylogeographic patterns of the coral reef sponge Pericharax heteroraphis in the south-west Pacific, focussing on the Great Barrier Reef (GBR). Variation among ITS sequences was low (<1.1% p-distance), in contrast to ATPSbeta-iII (<8.3% p-distance). Single-Stranded Conformation Polymorphism (SSCP) analysis proved to be an effective tool for phasing ATPSbeta-iII alleles of 292 bp length. Although sample sizes were limited for most populations and these results await corroboration by an extended sampling regime, a past population subdivision with subsequent range expansion was indicated by a ‘dumb-bell’ shaped statistical parsimony network of GBR ATPSbeta-iII alleles. Although no clear phylogeographic break was discovered on the GBR, the northern GBR was genetically differentiated from the central/southern GBR and Queensland Plateau, based on significant pairwise F _st values (0.137–0.275 and p ≤ 0.05) of pooled regional populations. The ATPSbeta-iII used in this study outperformed the frequently employed nrDNA ITS and might also turn out to be useful for phylogeographic studies of other coral reef taxa. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Integrative taxonomy of calcareous sponges (subclass Calcinea) from the Peruvian coast: morphology,molecules, and biogeography

Understanding of evolution and systematics of Calcarea (Porifera) have not yet met a corresponding increase in the knowledge of diversity and distribution of these sponges in several parts of the world. Peru is an emblematic example of this lack of taxonomic knowledge, as only three shallow‐water species of sponges have hitherto been reported from its 3000 km coast. With the aim of studying sponges of Peru, an integrative taxonomy approach (morphology, molecules, and biogeography) was used in order to achieve sound species identifications. The first findings of Peruvian calcareous sponges are presented here. Eight species are described in the subclass Calcinea, of which five are new to science. The retrieved biogeographical patterns are either locally endemic, widespread, or discontinuous over large areas. Clathrina antofagastensis was previously known from Chile, while C. aurea and Ernstia tetractina had been reported from the Atlantic (Brazil), and thus represent the first genetically confirmed tropical amphi‐American distributions of species not yet found on both sides of the Isthmus of Panama. Our results reveal a richer Tropical East Pacific sponge fauna than the Warm Temperate South‐Eastern Pacific one. © 2015 The Linnean Society of London     

Calcareous sponges of the genera Clathrina and Guancha (Calcinea, Calcarea, Porifera) of Norway (north-east Atlantic) with the description of five new species

The taxonomy and distribution of 11 species of calcareous sponges of the subclass Calcinea from the Norwegian coast are reviewed. The Norwegian Calcinea represents a mixture of southern boreal/boreal and boreoarctic species, and the calcinean sponge fauna of northern Norway has strong similarities to the Greenlandic and the White Sea/Barents Sea sponge faunas. Most Norwegian Calcinea have their main distribution between 20 and 100 m depth, although some species are found only in the shallow sublittoral or from sublittoral to abyssal depths. Six species were previously reported in the area: Clathrina coriacea (Montagu, 1818), Clathrina cribrata Rapp et al ., 2001, Clathrina nanseni (Breitfuss, 1896), Clathrina septentrionalis Rapp et al ., 2001, Guancha blanca Miklucho-Maclay, 1868 and Guancha lacunosa (Johnston, 1842). Five species are new to science: Clathrina corallicola , Clathrina jorunnae , Guancha arnesenae , Guancha camura , and Guancha pellucida spp. nov. A key to the known Norwegian Calcinea is provided.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 147 , 331–365.     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Genotype relationships inMicroseris elegans (Asteraceae,Lactuceae) revealed by DNA amplification from arbitrary primers (RAPDs)

The genetic relationships among 10 inbred lines representing 10 populations of the autogamous annualMicroseris elegans from throughout California has been determined using random amplified polymorphic DNAs (RAPDs). Seventeen arbitrary 10 base pair primers produced 134 amplification products; 81 of these were shared by two or more strains. The 3 genotypes from Northern California are closely related as are 3 genotypes from Middle Californian populations which are not nearest neighbors. DNA fingerprinting with the oligonucleotide (GATA)₄ gave compatible results, but the comparison was limited to samples run on one gel. Isoenzyme patterns are compatible with the DNA results, but limited by the very low number of informative polymorphisms. The clustered relationship among genotypes within a species and their geographic distribution suggests very restricted genetic recombination and an origin of new populations from randomly dispersed achenes within the range of the species.     

Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a species complex that is one of the most devastating agricultural pests worldwide and affects a broad range of food, fiber and ornamental crops. Unfortunately, using parsimony and neighbor joining methods, global phylogenetic relationships of the major races/biotypes of B. tabaci remain unresolved. Aside from the limitations of these methods, phylogenetic analyses have been limited to only small subsets of the global collection of B. tabaci, and thus limited taxon sampling has confounded the analyses. To improve our understanding of global B. tabaci phylogenetic relationships, a Bayesian phylogenetic technique was utilized to elucidate the relationships among all COI DNA sequence data available in GenBank for B. tabaci worldwide (366 specimens). As a result, the first well-resolved phylogeny for the B. tabaci species complex was produced showing 12 major well-resolved (0.70 posterior probability or above) genetic groups: B. tabaci (Mediterranean/Asia Minor/Africa), B. tabaci (Mediterranean), B. tabaci (Indian Ocean), B. tabaci (sub-Saharan Africa silverleafing), B. tabaci (Asia I), B. tabaci (Australia), B. tabaci (China), B. tabaci (Asia II), B. tabaci (Italy), B. tabaci (New World), B. tabaci (sub-Saharan Africa non-silverleafing) and B. tabaci (Uganda sweet potato). Further analysis of this phylogeny shows a close relationship of the New World B. tabaci with Asian biotypes, and characteristics of the major sub-Saharan Africa non-silverleafing clade strongly supports an African origin of B. tabaci due to its position at the base of the global phylogeny, and the diversity of well-resolved sub-clades within this group. Bayesian re-analyses of B. tabaci ITS, COI, and a combined dataset from a previous study resulted in seven major well-resolved races with high posterior probabilities, also showing the utility of the Bayesian method. Relationships of the 12 major B. tabaci genetic groups are discussed herein.     

The presence of the diagnostic character of the genus Paraleucilla (Amphoriscidae,Calcarea, Porifera) may depend on the volume and body wall thickness of the sponges

The main difference between the sponge genera Leucilla and Paraleucilla (Porifera, Calcarea, Amphoriscidae) is the presence of a disorganized zone (DZ) in the inner region of the skeleton of the latter genus. However, it has been repeatedly observed that specimens from different species of Paraleucilla lack this feature. It is assumed that the size of the sponge may have an effect on the presence or the absence of the DZ, but no investigation of this morphological variation has previously tested this hypothesis. Here, we examined this assumption and described the frequency with which the DZ is absent from individuals of Paraleucilla magna. We also investigated possible drivers of the observed variation using generalized linear models to evaluate whether the month of the year, rainfall, reproductive activity, volume, and body wall thickness could influence the presence or the absence of the DZ. The DZ was absent from 46.5% of the analyzed individuals, indicating that it may be misleading to use this trait to identify the genera Leucilla and Paraleucilla. The presence of the DZ in P. magna is influenced negatively by volume and positively by body wall thickness of the individuals. Our results confirm the previous assumptions for the family Amphoriscidae and highlight problems with the current classification of Calcarea. A discussion of the validity of some morphological characters and the importance of analyzing their variation is provided.