Nuclear 18S rDNA Research Articles

Five million years in the darkness: A new troglomorphic species of Cryptops Leach, 1814 (Chilopoda, Scolopendromorpha) from Movile Cave, Romania.

A new species of Cryptops Leach, 1814, C. speleorexsp. nov., is described from Movile Cave, Dobrogea, Romania. The cave is remarkable for its unique ecosystem entirely dependent on methane- and sulfur-oxidising bacteria. Until now, the cave was thought to be inhabited by the epigean species C. anomalans, which is widespread in Europe. Despite its resemblance to C. anomalans, the new species is well-defined morphologically and molecularly based on two mitochondrial (cytochrome c oxidase subunit I COI and 16S rDNA) and one nuclear (28S rDNA) markers. Cryptops speleorex sp. nov. shows a number of troglomorphic traits such as a generally large body and elongated appendages and spiracles, higher number of coxal pores and saw teeth on the tibia of the ultimate leg. With this record, the number of endemic species known from the Movile Cave reaches 35, which ranks it as one of the most species-rich caves in the world.

Integrative systematics reveals the new land-snail genus Taphrenalla (Eupulmonata: Ariophantidae) with a description of nine new species from Thailand

Abstract The endemic terrestrial Crown Snail from Southern Thailand formerly in Macrochlamys Gray, 1847 is now described as Taphrenalla Pholyotha & Panha gen. nov., based on comparative morphology and molecular data. Overall, eleven Taphrenalla species are now recognized, including two nominal species: T. asamurai and T. diadema. A total of nine new species are proposed: T. alba sp. nov., T. conformis sp. nov., T. corona sp. nov., T. dalli sp. nov., T. incilis sp. nov., T. macrosulcata sp. nov., T. parversa sp. nov., T. pygmaea sp. nov. and T. zemia sp. nov. The molecular phylogeny constructed from the mitochondrial cytochrome oxidase c subunit I (COI) and 16S rRNA gene fragments plus the nuclear 28S rDNA gene fragments revealed that Taphrenalla gen. nov. is monophyletic with a well-supported clade. The diagnostic characters of Taphrenalla gen. nov. are the shell sculpture with several radial grooves, body with well-developed colourful stripes running from the head to tail, and genitalia similar to Macrochamys but with an un-coiling epiphallic caecum. The spermatophore has one or two spines near the sperm sac and a spineless tail.

New pelagophytes show a novel mode of algal colony development and reveal a perforated theca that may define the class.

Pelagophytes (Heterokonta) are a morphologically diverse class of marine algae historically united only by DNA sequences. We established clonal cultures of sand-dwelling pelagophytes collected from intertidal pools around Australia. Phylogenetic trees based on nuclear 18S rDNA and plastid rbcL, psaA, psaB, psbA, and psbC sequences revealed two new genera, Gazia and Glomerochrysis, related to Aureoumbra in a distinct lineage within the Sarcinochrysidaceae (Pelagophyceae). The three new species (Gazia saundersii, Gazia australica, and Glomerochrysis psammophila), along with an Australian strain of Aureoumbra geitleri, are characterized by dominant benthic stages that differ significantly from one another, while occasionally producing classic heterokont zoospores. The benthic stage of Ga. saundersii has a novel development not observed in any other colonial alga, consisting of large, spherical colonies (up to 140μm in diameter) containing c. 2,500 cells that eventually differentiate and segregate into a large number of daughter colonies that are subsequently liberated. Alternatively, colonies may differentiate into a mass of zoospores that escape and settle to develop into new colonies. In Gl. psammophila, cubic packets of cells form large sticky clusters that bind sand together, while Ga. australica and A. geitleri are unicellular species. Using fixation by high-pressure freezing, a distinctive perforated theca was observed by TEM in all genera of this lineage, and we hypothesize this unique covering may be the first morphological feature to characterize most, if not all, pelagophytes. This study substantiates the diverse nature of sand-dwelling pelagophytes as well as their mechanisms for thriving in a dynamic habitat.

Molecular approach for analysis of in situ feeding by the dinoflagellate Noctiluca scintillans

The red tide forming heterotrophic dinoflagellate Noctiluca scintillans is common in temperate to tropical waters around the world. Understanding the in situ prey of N. scintillans is essential for elucidating its role in marine microbial food webs. In this study, we applied two polymerase chain reaction (PCR)-based cloning techniques, a predator-specific restriction enzyme, and a blocking primer. The PCR of nuclear 18S rDNA was performed on single N. scintillans cells that were collected from Ishinomaki Bay, Japan, in May 2018. The maximum detection rates of non-Noctiluca sequences were 56% using the restriction enzyme method and 87% with the blocking primer method, representing a broad taxonomic range of organisms, including diatom, dinoflagellate, bolidophyte, haptophyte, euglenophyte, green algae, golden algae, ciliate, heliozoa, copepod, brown seaweed, sponge, bivalve, and polychaete. The diverse DNA was probably ingested by N. scintillans directly or indirectly through secondary predation or ingestion of marine snow or detritus containing many organisms. The application of molecular approaches to various species may reveal undiscovered interactions within the phytoplankton community, including prey-predator, or symbiotic relationships.

Phylogeny of Adramini (Diptera, Tephritidae) based on integrative evidence

AbstractSpecies of the tribe Adramini (Tephritidae: Trypetinae) are usually slender, and some specific species have eyes borne at the ends of their long stalks. This tribe is mainly distributed in the tropics and subtropics of the Afrotropical, Oriental and Australasian Regions; relatively few species occur in the Palearctic and Nearctic Regions. The phylogeny of the tribe Adramini is presented here based on analysis of morphological and molecular information (DNA sequences of nuclear 28S rDNA, mitochondrial COI and COII, and 16S rDNA genes) for its representative species in most genera. Three monophyletic groups (Adrama‐com‐group, Pelmatops‐com‐group and Dimeringophrys‐com‐group) were discovered in the combined morphological and molecular tree. The results showed moderate support for the monophyly of Adramini and strong support for most of its genera. However, Euphranta appears to be polyphyletic. Sapadrama, Celidodacus and Euphranta are basal taxon, and Coelopacidia, Soita and Trypanophion are closely related to the stalk‐eyed fruit flies (Pelmatops + Pseudopelmatops). A hypothesis regarding the morphology–function relationships for two main groups of Adramini (Adrama‐group and Pelmatops‐group) with different evolving probabilities is inferred. Sapadrama is proposed be removed from Adramini; a new genus, Ichneumonomacula Chen gen. n. and a new species Ichneumonmacula wangyongi Chen sp. n., are recognized and described, and a key to recognize the genera of Adramini around the world is provided.

Genotyping ITS and matK regions of Hedera nepalensis K. Koch in Vietnam

Genotyping ITS and matK regions of Hedera nepalensis K. Koch in Vietnam

Scuttling towards monophyly: phylogeny of the mega‐diverse genus Megaselia (Diptera: Phoridae)

AbstractThe genus Megaselia Rondani (Diptera: Phoridae) is one of the largest in the animal kingdom, with nearly 1700 described species and many remaining to be discovered. Work on this group is notoriously challenging due to the extreme species diversity, poor knowledge of higher‐level relationships and lack of molecular data. In this paper, we present the largest study to date of Megaselia relationships based on molecular data from one nuclear (28S rDNA) and three mitochondrial (ND1, COI and 16S) markers for 175 Nordic specimens representing 145 species of Megaselia, plus outgroups. Based on phylogenetic analyses of these data, we propose 22 informal Megaselia species groups, all of which match well‐supported terminal clades. Relationships among these groups, and between them and several isolated species, remain largely uncertain. Of the 22, 20 species groups fall into a moderately well‐supported monophyletic clade of ‘core Megaselia’. Two species groups, the spinigera and ruficornis groups, fall outside of core Megaselia, as does the single representative of Myriophora, a genus that is included in Megaselia by some specialists. Here, we explore the morphology of these molecular species groups to aid future studies, and we discuss the implications of our findings for the generic circumscription of Megaselia. Hopefully, our results can aid further characterization of subgroups within the enormous Megaselia radiation and among its closest relatives, thus facilitating future work on this challenging but fascinating group of small flies.

Molecular Detection for Two Abalone Shell-boring Species Polydora haswelli and P. hoplura (Polychaeta, Spionidae) from Korea using 18S rDNA and cox1 Markers

Polydora species (Polychaeta: Spionidae) are morphologically similar and have shell-boring abilities that are harmful to commercially important mollusks; genetic analysis is therefore important for their identification. Here, molecular evidence for Polydora species in Korean cultured abalone (Haliotis discus hannai) is first reported. DNA barcode markers (Nuclear 18S rDNA and mitochondrial cox1 regions) identified Polydora haswelli and P. hoplura in infested abalone shells. The cox1 marker was efficient in detecting Polydora species even in samples contaminated with shell debris. The findings provide useful information for the accurate identification of Polydora species and for monitoring harmful spionids in mollusk aquaculture farms.

Phylogeography of the Marine Gastropod Reticunassa festiva Complex (Nassariidae) in the Coast of China

Revealing cryptic biodiversity and understanding their evolutionary histories is important to predict how climate change promoted diversification. The marine gastropod Reticunassa festiva is a common species distributed along the coast of China Seas and possesses cryptic diversity. In the present study, the phylogeographic pattern of R. festiva is investigated to test the potential mechanism that drove its diversification in China coastal regions using the mitochondrial cytochrome oxidase subunit I (COI) and nuclear 28S rDNA sequences. Three highly divergent COI clades (Clades A, B, and C) with considerable differences in genetic distances, haplotype diversity, and nucleotide diversity among each other were identified. The estimated divergence time between the two dominant clades (Clades A and B) was 2.0711 Ma, corresponding to the third major cool period in the Pleistocene. The sea surface temperature fluctuation in the subtropical ancient East China Sea could be the major drivers for the diversification of the two R. festiva clades. It is also suggested that the distribution pattern of Clades A and B is shaped by two major ocean currents. This study provides evidence for the correlation between species diversification and climate change in the NW Pacific during the Pleistocene.

Disentangling invasions in the sea: molecular analysis of a global polychaete species complex (Annelida: Spionidae: Pseudopolydora paucibranchiata)

The spionid polychaete Pseudopolydora paucibranchiata (Okuda, 1937) was originally described from Japan and has since been reported as a non-indigenous species in soft bottom communities in the Northeast Pacific, the Mediterranean Sea, around Europe, Australia, Brazil, and Florida. The diagnostic features of the adults are palps with ramified yellow chromatophores, prostomium rounded anteriorly, short occipital antenna on the caruncle, and a small disc-like pygidium. We collected Pseudopolydora with these features from locations worldwide and compared them by a molecular analysis. The Bayesian analysis of the combined dataset of three genetic markers (mitochondrial 16S rDNA, nuclear 28S rDNA and Histone 3; 811 bp in total) showed that the worms form a monophyletic group comprising four genetically different clades. We name this group the P. paucibranchiata complex and consider the clades as four pseudocryptic species. The largest examined clade comprises individuals from the Pacific Canada (British Columbia), Russia (Sea of Japan), South Korea (East Sea), Italy (Tyrrhenian and Ionian Seas), Australia (Victoria), Netherlands, and Japan, which we identify as P. paucibranchiata. The morphology, reproductive biology and ecology of P. paucibranchiata are briefly reviewed. The other three clades are referred to as Pseudopolydora vexillosa Radashevsky and Hsieh, 2000 (Vietnam, Nha Trang Bay), Pseudopolydora sp. A (Australia, Northern Territory), and Pseudopolydora sp. B (Kuwait, Arabian Gulf). We explain the occurrence of P. paucibranchiata outside of the Northwest Pacific by unintentional human-mediated transportation in ballast water and/or with commercial oyster movements in aquaculture operations, followed by successful invasions.

Bathy- and mesopelagic annelida from the Arctic Ocean: Description of new, redescription of known and notes on some “cosmopolitan” species

Pelagic polychaetes collected in the deep Arctic Ocean between 2011 and 2016 during three expeditions of R/V Polarstern were investigated using morphological taxonomic and molecular methods based on DNA sequences for portions of the mitochondrial 16S and nuclear 18S and 28S rDNA genes. A comparison of our results on arctic Pelagobia cf. longicirrata Greeff, 1879, (Lopadorrhynchidae) and Typhloscolex cf. muelleri Busch, 1851 (Typhloscolecidae) with the data on P. cf. longicirrata and T. cf. muelleri from the Atlantic, Antarctic and Pacific, suggests the presence of species complexes within each of them instead of cosmopolitan species. We infer the validity of Phalacrophorus borealis Reibisch, 1895 (Iospilidae), synonymized earlier by several authors with cosmopolitan P. pictus Greeff, 1879 as a subspecies. Also, we describe a new Lopadorrhynchidae genus, Bathypelagobia gen. nov. with a redescription of Pedinosoma polaris (Buzhinskaja, 2017). Finally, we describe a new Polynoidae species, Bathypolaria kondrashovi sp. nov. with some notes on the reproductive biology of the latter species.

Taxonomy and phylogeny of polypores with ganodermatoid basidiospores (Ganodermataceae)

Polypores with complex double-walled basidiospores with a pigmented endosporium ornamented with columns or ridges and a smooth hyaline exosporium are usually classified in the Ganodermataceae. Although the family presents well-delimited morphological features, recent phylogenetic studies have raised divergent evidences about its monophyly. In order to clarify uncertainties of generic delimitation and classification of the group and test its monophyly, morphological analysis and multilocus phylogenetic analyses based on Internal Transcribed Spacer, nuclear 28S rDNA, and portions of the largest subunit of RNA polymerase II and translation elongation factor 1-alpha were carried out. The main outcomes of the study are (i) Ganodermataceae is recovered as monophyletic, (ii) Amaurodermellus is proposed to accommodate the neotropical Amauroderma ovisporum, (iii) Cristataspora is proposed to accommodate two former species of Ganoderma - G. coffeatum and G. flaviporum - with pale context and truncate basidiospores with endosporic ornamentation as ridges. Comments on other species of Ganodermataceae are also presented.

Zealastoa Quicke & Ward, gen. nov., a new basal cyclostome braconid wasp (Hymenoptera: Braconidae) from New Zealand

AbstractThe Mesostoinae are an almost entirely Gondwanan subfamily of Braconidae, known from Australia, New Zealand and South America (extending into MesoAmerica). They are morphologically and biologically diverse and, together with Aphidiinae and Maxfischeriinae, are the sister group to all other extant cyclostome braconids. Here we describe and illustrate a new genus, Zealastoa Quicke & Ward, gen. nov., and three new species: Z. waitakerensis Quicke & Ward, sp. nov., Z. hirta Belokobylskij, Quicke & Ward, sp. nov., and Z. multiarticulata Belokobylskij, Quicke & Ward, sp. nov., all from New Zealand. Molecular phylogenetic analysis based on combined nuclear 28S rDNA D2‐D3 expansion region and the barcoding region of mitochondrial COI strongly support a monophyletic clade comprising the endemic New Zealand genera (Doryctomorpha Ashmead, Metaspathius Brues, Rhyssaloides and Zealastoa gen. nov.) as being the sister group to all other members of the subfamily.

Morphology and molecular phylogeny of Umbraulva spp. (Ulvales, Ulvophyceae), and proposal of Ryuguphycus gen. nov. and R. kuaweuweu comb. nov.

ABSTRACT The reproductive structures, early development and carotenoid composition of Umbraulva kuaweuweu, which is a deep-water ulvacean species recently reported from Hawaii, are reported for the first time using the specimens collected by dredging at ~30 m depth from Ogasawara Islands and Mageshima in southern Japan. Although Umbraulva kuaweuweu had siphonaxanthin as in U. japonica, a characteristic carotenoid of Umbraulva, U. kuaweuweu lacked α-carotene and β-carotene. U. kuaweuweu differed markedly from U. japonica in thallus morphology and reproductive structures: cell shape, presence/absence of inner rhizoidal filaments in the upper parts of thalli, solid/partly saccate thalli, and developmental processes of zoids. Multigene concatenated DNA sequences of chloroplast rbcL and tufA genes, and nuclear 18S, 5.8S, 28S rDNA and rDNA ITS1 and ITS2 regions indicated separation of U. kuaweuweu from the clade comprised of the generitype U. japonica, U. amamiensis, U. dangeardii and U. kaloakulau. In conclusion, based on the distinctive morphology, life history, carotenoid composition and molecular phylogeny, we propose the establishment of a new genus Ryuguphycus gen. nov. to accommodate U. kuaweuweu, as R. kuaweuweu comb. nov.

Interfamilial relationships in order Fabales: new insights from the nuclear regions sqd1 and 26S rDNA

Leguminosae, Polygalaceae, Quillajaceae and Surianaceae together comprise the order Fabales. Phylogenetic relationships within Fabales remain an unsolved problem even though interfamilial relationships have been examined in a number of studies using different sampling approaches and both molecular and morphological data. In this study, we gather information from the nuclear 26S rDNA region as well as previously published data from the sqd1, matK and rbcL regions. Phylogenetic analyses were performed by maximum parsimony, maximum likelihood and Bayesian inference. Overall, the best-supported topology for the relationships among families within the order places the pair of Leguminosae and Polygalaceae as sister to the pair of Quillajaceae and Surianaceae. However, our approximately unbiased (AU) test of the combined data results has shown that none of the seven different topologies rejected. Furthermore, three topologies were not significantly different from each other. Therefore, similar to the previous studies, this study did not find well-supported dichotomous relationships among the four Fabales families. The Fabales topology was very sensitive to both data choice and the phylogenetic methods used, which may indicate a rapid-near-simultaneous evolution of the four Fabales families. Our results also show that while nuclear sqd1 can be helpful as a complementary region, both the nuclear sqd1 and rDNA 26S regions could be problematic when analyzed individually.

A new species rises from beneath Florida: molecular phylogenetic analyses reveal cryptic diversity among the metapopulation of Crangonyx hobbsi Shoemaker, 1941 (Amphipoda: Crangonyctidae)

The phylogenetic structure of 23 populations of Crangonyx hobbsi, a stygobitic amphipod species endemic to the Floridan aquifer, is detailed. Molecular genetic analyses (based on three genes the nuclear 18S rDNA, 28S rDNA, and mitochondrial 16S rDNA) point to the presence of at least two phylogenetic clades within the aquifer. These clades align into two major zoogeographic regions, the Northern Suwannee River Basin and the Coastal and Central Lowlands in the Florida peninsula. Three species delimitation models indicate that the Northern Suwannee River Basin Clade represents a species level differentiation from C. hobbsi sensu stricto. Morphological analyses support the molecular analyses, revealing the presence of several taxonomically significant differences between the two species, including presence/absence of calceoli on the second antennae of males and differences in telson shape in both sexes. Additionally, the molecular diversity of C. hobbsi sensu stricto was shown to be surprisingly variable, suggesting the potential presence of additional diversity within the taxon.

Identification of Spruce Budworm Natural Enemies Using a qPCR-Based Molecular Sorting Approach

Annual monitoring of mortality agents in the course of a spruce budworm (Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)) population cycle is essential to understanding the factors governing the rise and collapse of outbreaks. To date, assessments of causes of budworm mortality have relied on laboratory rearing of field-collected larvae, followed by visual identification of emerging parasitoids and/or microscopic analysis of pathogens in larval carcasses. Although this approach has provided vital information on the abundance and identity of mortality agents, the procedure is labor-intensive and has limits in terms of accuracy. To overcome these shortcomings, we developed a molecular identification tool that makes use of real-time quantitative PCR (qPCR) and TaqMan® technologies. The tool relies on taxon-specific molecular variants (single nucleotide polymorphism [SNP] markers) found in mitochondrial (COI) and nuclear (28S rDNA) genes, for parasitoids, and in the nuclear SSU rDNA gene for microsporidian pathogens; these are then used as molecular signatures targeted by qPCR primers and TaqMan probes. Thus, the design of several sets of primers and probes deployed in multiplex format enables the identification of natural enemies via a molecular sorting process, bypassing barcode sequencing. Crude budworm DNA extracts are processed through a first module that detects dipteran and hymenopteran parasitoids, and microsporidian infections. Positive samples are then processed for species determination using three additional modules, enabling the identification of 20 common natural enemies of the spruce budworm. The tool has been fully validated using DNA samples from all comprised taxa, and both its sensitivity and accuracy compared favorably with the rearing-based method in an analysis of field-collected budworms. Using this tool, sample processing can be completed within two days, does not require larval rearing, provides accurate species identification, and can be conducted by technical staff without extensive molecular biology or insect taxonomy training.

Multivariate ratio analysis and DNA markers reveal a new Australian species and three synonymies in eucalypt-gall-associated Megastigmus (Hymenoptera: Megastigmidae).

The genus Megastigmus Dalman, 1820 (Hymenoptera: Megastigmidae) contains potential biocontrol agents of the invasive eucalypt galling chalcid Leptocybe spp. (Hymenoptera: Eulophidae), with several species reported in various parts of the world. Species discrimination is challenging due to intraspecific morphological variation, difficulty in measuring sizes of body parts, and the lack of information regarding the global distribution of parasitic Megastigmus. We used two species commonly associated with Leptocybe in its native range to review taxonomic methods and determine the most reliable morphological characters in species delimitation. We examined size variation of body characters, and conducted species discrimination using multivariate ratio analysis, mitochondrial Cytochrome c oxidase subunit 1 (COI) and nuclear 28S rDNA (28S) sequences. Morphological traits were effective in species delimitation yet revealed high variation in several characters employed in current keys. Knowledge generated on morphology and DNA justified the description of a new species, M. manonae, sp. n., the first record of M. pretorianensis in Australia, and revised diagnostic characters for M. zvimendeli. Based on these diagnostic characters and molecular data, we synonymize three species (M. judikingae, syn. n., from Australia, M. sichuanensis, syn. n., from China and M. icipeensis, syn. n., from Kenya) with M. zvimendeli. Our findings highlight the importance of molecular markers in assisting taxonomic decision-making and the need for coordinated work in identifying Megastigmus associated with Leptocybe spp.

Distribution of 18s rDNA variants of Diderma hemisphaericum (Myxomycota) specimens obtained from moist chambers of materials collected in the Philippines and Vietnam

Since there are only limited biogeographic studies of myxomycetes in Southeast Asia, this study was conducted to assess the molecular diversity of Diderma hemisphaericum, a cosmopolitan myxomycetes species that abundantly occurs in most moist chamber collections in tropical Southeast Asia. Specimens that contain D. hemisphaericum were gathered from various parts of the Philippines: (i) Los Banos, (ii) Calauan, (iii) Basud, (iv) Isarog and (v) Malilipot; and Vietnam: (i) Thai Nguyen (ii) Ha Noi and (iii) Da Nang. Using modern molecular techniques, the common barcode nuclear marker 18S rDNA for myxomycetes was targeted for the 50 specimen used in this study. A total of 27 ribotypes (11 from the Philippines and 16 from Vietnam) were obtained. The Mantel test showed that genetic differentiation among ribotypes is not correlated with geographic distances. The FST index (0.083) indicates high genetic intermixing between the Philippines and Vietnam. Hence, it seems that the variation across regional scale gene pool is not directly influenced by geographic isolation but rather by environmental selection.

Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world. The morphological, physiological, and biochemical characteristics are different among geographic strains, which can not be distinguished with nuclear ribosomal DNA markers at present. Therefore, the genetic distance and phylogeographic relationships of nuclear 28S rDNA D1-D2 and ITS regions, and three chloroplast intergenic spacers (petN-trnS1, trnM1-psbA, and rbcS-rpl27) were analyzed and compared among 13 strains of P. globosa isolated from the Pacific Ocean and Atlantic Ocean in this study. In addition, the nucleotide polymorphisms of 28S rDNA D1-D2, ITS, and rbcS-rpl27 regions were evaluated in two P. globosa strains. The various levels of nucleotide polymorphism were in the nuclear 28S rDNA D1-D2 region and ITS region, but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer. A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer, which had the strongest distinction to geographic strains compared to those of 28S rDNA D1-D2 and ITS regions. In the phylogenetic tree of rbcS-rpl27 intergenic spacer, the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P. globosa, and then formed an independent clade, while the other Atlantic strains and all of Pacific strains joined up to build the other clade. It was implied that at least two genetically distant populations of P. globosa existed in the Atlantic coastal regions. This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P. globosa, and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P. globosa.