Nuclear 18S rRNA Gene Research Articles

Molecular evidence resolving the confusion of two species of Spilopteron (Hymenoptera: Ichneumonidae) caused by marked geographical colour variation

The delimitation of two ichneumonid species, Spilopteron apicale (Matsumura) and S. tosaense (Uchida), was investigated using DNA sequences of the mitochondrial COI and nuclear 28S rRNA genes, as well as adult morphology. The two species have long been confused and were until recently speculated to be the same species with continuous colour variation. Our molecular and mor- phological studies reveal that there are two distinct species: a dark species confined to northern or high latitude localities (S. apicale ) and a widely distributed species with a marked geographical gradient in body colour (S. tosaense) across the Japanese Archipelago. In the latter species, female body colour became significantly darker with latitude and altitude. A lectotype of Chorischizus apicalis Matsumura, 1912 is designated.

Phylogenetic position of Loricifera inferred from nearly complete 18S and 28S rRNA gene sequences.

BackgroundLoricifera is an enigmatic metazoan phylum; its morphology appeared to place it with Priapulida and Kinorhyncha in the group Scalidophora which, along with Nematoida (Nematoda and Nematomorpha), comprised the group Cycloneuralia. Scarce molecular data have suggested an alternative phylogenetic hypothesis, that the phylum Loricifera is a sister taxon to Nematomorpha, although the actual phylogenetic position of the phylum remains unclear.MethodsEcdysozoan phylogeny was reconstructed through maximum-likelihood (ML) and Bayesian inference (BI) analyses of nuclear 18S and 28S rRNA gene sequences from 60 species representing all eight ecdysozoan phyla, and including a newly collected loriciferan species.ResultsEcdysozoa comprised two clades with high support values in both the ML and BI trees. One consisted of Priapulida and Kinorhyncha, and the other of Loricifera, Nematoida, and Panarthropoda (Tardigrada, Onychophora, and Arthropoda). The relationships between Loricifera, Nematoida, and Panarthropoda were not well resolved.ConclusionsLoricifera appears to be closely related to Nematoida and Panarthropoda, rather than grouping with Priapulida and Kinorhyncha, as had been suggested by previous studies. Thus, both Scalidophora and Cycloneuralia are a polyphyletic or paraphyletic groups. In addition, Loricifera and Nematomorpha did not emerge as sister groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-015-0017-0) contains supplementary material, which is available to authorized users.

Taxonomic Resolutions Based on 18S rRNA Genes: A Case Study of Subclass Copepoda

Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1–9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

Molecular phylogeny of commercially important lobster species from Indian coast inferred from mitochondrial and nuclear DNA sequences

Lobsters constitute low-volume high-value crustacean fishery resource along Indian coast. For the conservation and management of this declining resource, accurate identification of species and larvae is essential. The objectives of this work were to generate species-specific molecular signatures of 11 commercially important species of lobsters of families Palinuridae and Scyllaridae and to reconstruct a phylogeny to clarify the evolutionary relationships among genera and species included in this study. Partial sequences were generated for all the candidate species from sampling sites along the Indian coast using markers like Cytochrome oxidase I (COI), 16SrRNA, 12SrRNA, and 18SrRNA genes, and analyzed. The genetic identities of widely distributed Thenus species along the Indian coast to be Thenus unimaculatus and the sub-species of Panulirus homarus to be P. homarus homarus were confirmed. Phylogeny reconstruction using the individual gene and concatenated mtDNA data set were carried out. The overall results suggested independent monophyly of Scyllaridae and Stridentes of Palinuridae. The interspecific divergence was found to be highest for the 12SrRNA compared with other genes. Significant incongruence between mtDNA and nuclear 18SrRNA gene tree topologies was observed. The results hinted an earlier origin for Palinuridae compared with Scyllaridae. The DNA sequence data generated from this study will aid in the correct identification of lobster larvae and will find application in research related to larval transport and distribution.

Vicariance ofPyrocoeliafireflies (Coleoptera: Lampyridae) in the Ryukyu islands, Japan

We performed molecular phylogenetic analyses based on the mitochondrial COI gene (687 bp) and the nuclear 28S rRNA gene (715 bp) and reconstructed phylogenetic trees of the Pyrocoelia fireflies in the Ryukyu Islands and eastern Asia. Age calibration was done using a robust geological constraint: the Okinawa trough and associated straits began to rift at 1.55 Ma, isolating the Ryukyu Islands from the Chinese continent, Japanese islands, Taiwan island and some of the islands from each other. We suggest that the physical isolation of these islands began to generate the allopatric speciation within these islands, so the timing of this isolation was assigned to an appropriate node. The topology is completely concordant among phylogenetic trees reconstructed using MEGA (maximum-likelihood), raxmlGUI (maximum-likelihood) and BEAST (Bayesian inference; including combined analysis of COI and 28S rRNA genes). Two lineages are recognized, related to their emergence time; spring to summer, and autumn. In each lineage, vicariance is inferred to have begun at 1.55 Ma from our phylogenetic and geological analyses. In lineage 1, P. oshimana (Amami), P. matsumurai (Okinawa), P. discicollis (W. Japan), P. fumosa (E. Japan) and P. abdominalis (Yaeyama) were differentiated. In lineage 2, P. rufa (Tsushima and Korea), P. miyako (Miyako-jima), P. atripennis (Ishigaki-jima) and P. praetexta (Taiwan and HongKong) were differentiated. Pyrocoelia analis (Taiwan and China) emerges throughout the year except for winter, and constitutes another lineage. We suggest that Pyrocoelia fireflies differentiated at 2 Ma to generate these three lineages. The base substitution rate for the COI gene is estimated as 4.48% Myr –1 and that for the 28S rRNA gene is 0.394% Myr –1 , and these rates were used in a combined BEAty analysis in BEAST. © 2015

Runcinida valentinae Chernyshev, 2006 (opisthobranchia: Runcinacea), a new opisthobranch species for the Sea of Japan

Runcinida valentinae, an opisthobranch mollusk that was previously known from the waters off Iturup Island (Kuril Islands), has been found off Rudnaya Bay and in Oprichnik Bay in the Sea of Japan. New data on the radula morphology in this species are presented. Partial sequences of nuclear 28S rRNA and mitochondrial COI genes of the genus Runcinida have been obtained for the first time. Their analysis shows the distinctiveness of R. valentinae, which is morphologically close to R. marisae Chernyshev, 1998 from Peter the Great Bay. The strong nucleotide differences between these species confirm importance of body coloration patterns as species-specific traits in runcinid systematics.

An integrative taxonomic investigation of the diversity of digenean parasites infecting the intertidal snail Austrolittorina unifasciata Gray, 1826 (Gastropoda: Littorinidae) in Australia.

We investigated for the first time the digenean parasites of Austrolittorina unifasciata Gray (Gastropoda: Littorinidae), a periwinkle snail inhabiting the rocky shores of Australia. Here we present detailed morphological descriptions and molecular data (sequences for the mitochondrial cox1 and the nuclear 28S rRNA gene) for the cercariae and intramolluscan stages of the digenean parasites found. Five species, one each of the families Notocotylidae Lühe, 1909, Gorgocephalidae Manter, 1966 and Philophthalmidae Looss, 1899, and two of the family Renicolidae Dollfus, 1939, were recorded and characterised molecularly. Phylogenetic analyses at the superfamily level provided evidence for the familial and generic affiliation of the species and their relationships with congeners. This study is the first to provide data on the life cycle of a species of the family Gorgocephalidae, a parasite of kyphosid fish for which only adult stages had, thus far, been described. The relatively high prevalence of this species allowed mapping of the cox1 haplotype distribution of Gorgocephalus sp. Aus along the southern coast of New South Wales.

28S Ribosomal RNA Sequences Separate Five Prominent Lygus (Hemiptera: Miridae) Pest Species Into Three Species Clusters

ABSTRACT A segment of the nuclear 28S rRNA gene was compared among six species of Lygus [Lygus hesperus (Knight), Lygus keltoni (Schwartz), Lygus borealis (Kelton), Lygus elisus (Van Duzee), Lygus lineolaris (Palisot de Beauvois), and Lygus vanduzeei (Knight)]. The DNA sequences were separate into three main groups. The LL group contains L. lineolaris and L. vanduzeei. Group LBLE is composed of L. elisus and most of L. borealis. Group LH includes L. hesperus and most of L. keltoni. Some L. keltoni were part of the LBLE group and some L. borealis were part of the LH group. The 28S region does not contain sufficient genetic polymorphism to delineate species. The apparent polyphyly of L. borealis and L. keltoni could reflect historic interbreeding, recent development of a hybrid swarm, or highlight inadequacies of morphospecies identification.

A new species of Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae) from the double-crested cormorant Phalacrocorax auritus (Lesson) (Aves: Phalacrocoracidae) in North America.

Drepanocephalus auritus n. sp. is described based on specimens from the double-crested cormorant Phalacrocorax auritus (Lesson) in North America. The new species differs from its congeners in its very narrow, elongate body, long uterine field and widely separated testes. Sequences of the nuclear rRNA gene cluster, spanning the 3' end of the nuclear ribosomal 18S rRNA gene, internal transcribed spacer region (ITS1+5.8S gene+ITS2) and partial 28S gene (2,345 bp), were identical in specimens collected from North Dakota, Minnesota and Mississippi, USA. Sequences of the 651 bp long fragment of the mitochondrial cox1 gene exhibited very low intraspecific variability (< 1%). Comparisons of the newly-generated sequences with those available in the GenBank indicate that the sequences from North America published under the name D. spathans Dietz, 1909 in fact represent D. auritus n. sp.

Molecular phylogeny of Panorpidae (Insecta: Mecoptera) based on mitochondrial and nuclear genes

Panorpidae are the largest family in Mecoptera, covering approximately 70% species of the order. However, the phylogenetic relationship within Panorpidae has not been adequately explored. Here we analyzed the phylogenetic relationships among 70 species of five genera in Panorpidae using maximum likelihood and Bayesian inference based on two mitochondrial (cox1 and cox2) and one nuclear (28S rRNA) gene fragments with Panorpodes kuandianensis and Brachypanorpa carolinensis in Panorpodidae as outgroups. The results show that the genera Neopanorpa, Sinopanorpa and Dicerapanorpa are monophyletic, while the widespread genus Panorpa is reconfirmed to be a paraphyletic group. The P. centralis group is monophyletic and may merit a generic status, while the P. davidi and P. amurensis groups are paraphyletic. The divergence time estimated from BEAST analysis indicates that the Panorpidae may originate in the period from early Paleogene (63.6mya) to middle Eocene (41.2mya), and most diversification within Panorpidae occurred in the Cenozoic. The phylogeny and biogeography of Panorpidae are briefly discussed.

Phylogenetic Position of the Queer, Backward-bent Entoproct Loxosoma axisadversum (Entoprocta: Solitaria: Loxosomatidae)

The solitary entoproct Loxosoma axisadversum Konno, 1972 was found on the body surface of the maldanid polychaete Nicomache personata Johnson, 1901 obtained among roots of the seagrass Phyllospadix iwatensis Makino collected at a depth of about 1 m in Oshoro Bay, Hokkaido, Japan. The species had previously been known from elsewhere in northern Japan, attached to another maldanid, Nicomache minor Arwidsson, 1907. The morphology of the new material is briefly described with photographs taken in life. A preliminary phylogenetic analysis using concatenated partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene, as well as the nuclear 18S and 28S rRNA genes, placed L. axisadversum as the sister taxon to all the rest of Solitaria included in the analysis, indicating non-monophyly of the genus Loxosoma Keferstein, 1862.

Redescription and phylogenetic relationships of Euparyphium capitaneum Dietz, 1909, the type-species of Euparyphium Dietz, 1909 (Digenea: Echinostomatidae).

Euparyphium capitaneum Dietz, 1909, the type-species of the genus Euparyphium Dietz, 1909, is described on the basis of material collected from the type-host Anhinga anhinga (L.) from Pascagoula River, which drains into the northern coast of the Gulf of Mexico. Combination of light and scanning electron microscopy observations of freshly collected and properly fixed specimens in our study has allowed us to provide novel information on the morphology and topology of the reproductive systems and other morphological features of the species. A Bayesian inference analysis based on the newly-obtained partial sequence of the nuclear 28S rRNA gene for E. capitaneum and 24 previously published sequences from the superfamily Echinostomatoidea Looss, 1899 provided evidence supporting the distinct status of the genera Euparyphium and Isthmiophora Lühe, 1909.

New Zealand screw shells Maoricolpus roseus (Gastropoda: Turritellidae): two species, two subspecies or a single variable species?

The turritellid Maoricolpus roseus (Quoy & Gaimard, 1834) is abundant in low intertidal and shallow water marine environments around the coasts of New Zealand. It is currently divided into two regional subspecies: M. roseus roseus, common around much of New Zealand's coast; and M. roseus manukauensis, restricted to several west coast harbours in the North Island, specifically Manukau, Raglan and Kawhia. Molecular data, consisting of both mitochondrial (16S rRNA and cytochrome oxidase I (COI)) and nuclear (18S rRNA and 28S rRNA) genes, were collected and analysed to resolve the status of these subspecies. Our analyses reveal that neither subspecies is monophyletic in our phylogenetic trees, and that the genetic distances between them are no greater than those within. We thus argue that M. roseus manukauensis should be synonymised with the nominotypical subspecies. Maoricolpus roseus is a morphologically variable species, with low levels of 16S and COI genetic diversity within and among different populations.

Allopatry and overlap in a clade of snails from mangroves and mud flats in the Indo-West Pacific and Mediterranean (Gastropoda: Potamididae:Cerithideopsilla)

Cerithideopsilla is a genus of potamidid snails found in high abundance on sedimentary intertidal flats and beneath mangrove trees on continental shores in the tropical and subtropical Indo-West Pacific region and Mediterranean Sea. Taxonomic revisions have recognized four species, but recent molecular studies have hinted at a higher diversity. Here, we analyse 377 individuals sampled from across the known range and use a combination of molecular phylogenetic (mitochondrial COI and 16S rRNA, and nuclear 28S rRNA genes), statistical (generalized mixed Yule-coalescent GMYC method) and morphological (shell form) criteria to delimit 16 species. These form four species groups, corresponding with the traditionally recognized species C. alata, C. ‘djadjariensis’ (for which the valid name is C. incisa), C. cingulata and C. conica. Distribution maps were compiled using museum specimens identified by diagnostic shell characters. In combination with the molecular phylogenetic trees, these suggest an allopatric speciation mode, with diversification centred on the East Asian coastline and northern Australia, and a pronounced gap in the ‘eastern Indonesian corridor’, an area of low oceanic productivity. There is, however, frequently geographical overlap between sister species and we suggest from several sources of evidence (e.g. presence of C. conica in isolated saline lakes 900 km from the sea) that post-speciation transport by migratory birds has occurred. Nine of the 16 species occur between the Gulf of Tonkin and Hong Kong, so southern China is significant for both the evolution and conservation of Cerithideopsilla species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114, 212–228.

Montenegrospeum Pešić et Glöer, 2013: a representative of moitessieriidae?

The paper deals with phylogenetic relationships of recently described Montenegrospeum bogici Pesic et Gloer, 2012 from central Bosnia. The female reproductive system was found to bear two receptacula seminis. Mitochondrial cytochrome oxidase subunit I and nuclear 18S rRNA gene partial sequences were used for the maximum likelihood phylogenetic inference. Morphological and molecular data are congruent: Montenegrospeum does not belong to the Moitessieriidae, and is phylogenetically remote from Bythiospeum; it belongs to the Hydrobiidae, subfamily Sadlerianinae, and its sister taxon is Dalmatinella Radoman, 1973 (bootstrap support 87%). key words: Truncatelloidea, Moitessieriidae, Sadlerianinae, female reproductive organs, COI, 18S rRNA, molecular phylogeny

An effective procedure for DNA isolation and voucher recovery from millimeter-scale copepods and new primers for the 18S rRNA and cytb genes

Many investigators need to determine whether individuals belong to the same species. DNA-sequence data have helped with this task, but current procedures of DNA isolation from millimeter-scale crustaceans, such as harpacticoid copepods, leave little to no voucher material for morphological analysis, and many procedures yield only enough DNA for a single amplification reaction. We therefore developed a DNA-isolation procedure that yielded essentially intact exoskeletons and sufficient DNA for multiple polymerase chain reactions.DNA-amplification success of our DNA-isolation procedure was relatively insensitive to (1) the length of preservation time from sample collection to DNA isolations and (2) the length of time the DNA was stored at −20°C after isolation. An additional benefit of our procedure is therefore that the DNA isolated is relatively stable.Primers available for the nuclear 18S rRNA gene and the mitochondrial cytochrome oxidase b (cytb) gene are known not to work for many harpacticoids. We therefore designed primers that would amplify and sequence an ~750-base-pair fragment of the 18S rRNA gene and others that would amplify and sequence an ~450-base-pair fragment of the cytb gene. Both primer sets worked for at least 12 harpacticoid families.

Molecular phylogeny of Orthetrum dragonflies reveals cryptic species of Orthetrum pruinosum.

Dragonflies of the genus Orthetrum are members of the suborder Anisoptera, family Libellulidae. There are species pairs whose members are not easily separated from each other by morphological characters. In the present study, the DNA nucleotide sequences of mitochondrial and nuclear genes were employed to elucidate the phylogeny and systematics of Orthetrum dragonflies. Phylogenetic analyses could not resolve the various subfamilies of the family Libellulidae unequivocally. The nuclear 28S rRNA gene is highly conserved and could not resolve congeneric species of Orthetrum. Individual mitochondrial genes (COI, COII, and 16S rRNA) and combination of these genes as well as the nuclear ITS1&2 genes clearly differentiate morphologically similar species, such as the reddish species pairs O. chrysis and O. testaceum, and the bluish-coloured species O. glaucum and O. luzonicum. This study also reveals distinct genetic lineages between O. pruinosum schneideri (occurring in Malaysia) and O. pruinosum neglectum (occurring north of Peninsular Malaysia from India to Japan), indicating these taxa are cryptic species.

Description of a new species of Sternomoera (Crustacea: Amphipoda: Pontogeneiidae) from Japan, with an analysis of the phylogenetic relationships among the Japanese species based on the 28S rRNA gene.

A new species of amphipod, Sternomoera morinoi Tomikawa and Ishimaru, is described from subterranean aquatic habitats in Shiga Prefecture, Japan. In addition, Relictomoera tsushimana (Uéno, 1971) from a well on the island of Tsushima in Japan is transferred to Sternomoera and redescribed based on the holotype. Sternomoera morinoi sp. nov. is most similar to S. tsushimana (Uéno, 1971) comb, nov., but is distinguished by having many fine setae on the body, a shorter antenna 1, fewer C-setae on mandibular palp article 3, a shorter mandibular palp article 3, sparsely setose anterior margins of coxae 1-4, a different armature of the palmar margins of gnathopods 1 and 2, and no long setae on the posterior margin of the basis in gnathopod 2 and pereopods 3 and 4. The phylogenetic relationships among the Japanese species of Sternomoera are also estimated, based on partial DNA sequences of the nuclear 28S rRNA gene.

Amblyomma hadanii n. sp. (Acari: Ixodidae), a tick from northwestern Argentina previously confused with Amblyomma coelebs Neumann, 1899.

All stages of Amblyomma hadanii n. sp. (Acari: Ixodidae) are described from northwestern Argentina. The diagnostic characters for males are a combination of the pattern of scutal ornamentation, basis capituli dorsally rectangular with cornua, coxa I with two subequal spurs (the internal wider, the external longer), coxae II-III with a single spur, coxa IV with a single spur not reaching level of anus, ventral plates irregular in shape (larger and sometimes with a small incision on festoons 4, 5 and 6) and hypostome spatulate with dental formula 3/3 in 7-8 rows. The diagnostic characters for the females are a combination of scutal ornamentation, postero-lateral margins of scutum slightly convex, coxa I with two subequal spurs (the internal wider, the external longer), basis capituli dorsally rectangular, porose areas rounded, genital aperture U-shaped, and hypostome spatulate with dental formula 3/3 in 7-8 rows. Diagnosis of nymphs can be performed by a combination of basis capituli rectangular, scutum with large punctations in the lateral fields and small punctations in the central field, and cervical groove short and ending as a small shallow depression at the eye level. Larvae are diagnosed by the shape of basis capituli, scutum with with posterior margin slightly convex, and legs with coxa I with 2 triangular spur (the external longer than the internal), and with coxae II and III each with 1 triangular spur. The hosts recorded for this new tick species are Tapirus terrestris (Linnaeus), horse, cattle, dog and humans. Analyses of a 410 bp fragment of the mitochondrial 16S rRNA gene and the complete sequence of the nuclear 18S rRNA gene supported the description of A. hadanii as a new species.

Occurrence of true branches in Rhizoclonium (Cladophorales, Ulvophyceae) and the reinstatement of Rhizoclonium pachydermum Kjellman

The phylogenetic position of the freshwater green alga Rhizoclonium pachydermum (Ulvophyceae: Cladophorales) was investigated using nuclear 18S rRNA gene and internal transcribed spacer 2 (ITS2) sequences. This alga has been referred to as Cladophora pachyderma. Based on its morphology, it was formerly classified in the section Affines in the genus Cladophora. However, this classification was not supported by the current phylogenetic analyses, where Rhizoclonium pachydermum formed a well-supported clade with other Rhizoclonium species. We consider that Rhizoclonium possesses real branches and the most important criteria that characterize the genus are: long unbranched filaments only with rhizoid branches, or only branched at the basal region of the thallus; and cylindrical cells with few or limited numbers of nuclei.