Partial DNA Sequence Analysis Research Articles

A new species of freshwater snail of Fenouilia (Gastropoda, Pomatiopsidae) from northern Guangxi, China, based on morphological and DNA evidence.

A new species of pomatiopsid freshwater snail, Fenouiliaundata Chen & He, sp. nov., is described from Guangxi, China, based on morphological and molecular evidence. The new species can be distinguished from its congeners by the following combination of characters: shell with low, prosocline, rounded axial ribs and fine spiral striae, broader than high; aperture broader than shell height; radula with lateral teeth have only two or three faint, wavy ridges on inner side. A molecular analysis of partial mitochondrial COI and 16S DNA sequences supports the systematic position of the new taxon.

Further insight into the genetic diversity of Entamoeba coli and Entamoeba hartmanni.

Despite the species' wide distribution, studies of the genetic diversity within Entamoeba coli and Entamoeba hartmanni remain limited. In the present study, we provide further insight into the genetic diversity of both species based on analysis of partial nuclear small subunit ribosomal DNA sequences generated from human fecal DNAs from samples collected in Africa, South America, and Europe. Reinforcing the previous recognition that E. coli is a species complex, our data confirm the existence of the two subtypes, ST1 and ST2, previously identified plus, potentially, a new subtype, ST3. While ST1 appears to be genetically quite homogenous, ST2 shows a substantial degree of intrasubtype diversity. ST2 was more common in samples collected outside Europe, whereas ST1 showed no geographical restriction. The potentially novel subtype is represented to date exclusively by sequences from South American and African samples. In contrast to previous reports, our new data also indicate substantial variation in E. hartmanni that could also support the establishment of subtypes within this species. Here, however, no links were identified between subtype and geographical origin.

Prevalence and diversity of avian haemosporidian parasites across islands of Milne Bay Province, Papua New Guinea

The taxonomically diverse and relatively understudied avifauna of Papua New Guinea’s (PNG) island archipelagos provide a unique ecological framework for studying haemosporidian parasite differentiation and geographic structure. We implemented molecular and phylogenetic analyses of partial mitochondrial DNA sequences to assess the host distribution of 3 genera of vector-transmitted avian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) across a range of islands off the southeastern tip of PNG. We identified 40 new lineages of haemosporidians, including five lineages belonging to Leucocytozoon, a genus not previously described in this region. Leucocytozoon infections were only observed on the larger, human-inhabited islands. Lineages belonging to Haemoproteus were diverse and had broad geographic distribution. Compared to the mainland, Haemoproteus parasites on the smaller, more distant islands had greater host specificity and lower infection prevalence. The black sunbird (Leptocoma aspasia), a commonly caught species, was shown to be a rare host for Haemoproteus spp. infections. Moreover, although birds of the genus Pitohui harbor a neurotoxin (homobatrachotoxin), they demonstrated an infection prevalence comparable to other bird species. The islands of PNG display heterogeneous patterns of haemosporidian diversity, distribution and host-specificity and serve as a valuable model system for studying host-parasite-vector interactions.

Neoveronaea sinensis gen. & sp. nov. from Jiangxi, China

A new hyphomycete genus and species, Neoveronaea sinensis, is described from Jiangxi Province, southern China. Neoveronaea is characterized by its macronematous conidiophores, and euseptate, obovoid to ellipsoidal, pale brown, smooth conidia. Phylogenetic analyses of partial DNA sequences of internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU), using Maximum-Likelihood and Bayesian Inference, reveal the taxonomic placement of Neoveronaea within the Herpotrichiellaceae, in which it forms a lineage distinct from other genera.

Population genetic structure and dispersal patterns of grey wolfs (Canis lupus) and golden jackals (Canis aureus) in Georgia, the Caucasus

AbstractGrey wolf and golden jackal are both common in Georgia, although they have different habitat preferences. The wolf is more common in mountain areas of the country, and jackals are more common in the lowland part of Georgia, with its milder and warmer climate. In recent decades, the abundance of both species increased. Simultaneously, the jackals are increasingly often sighted at higher elevations than previously recorded, and simultaneously, there are increased sightings of the wolves in lowlands of western Georgia, including the areas close to the Black Sea Coast. The analysis of partial mitochondrial DNA sequences and 20 microsatellite markers suggest substantially higher genetic diversity of wolves than the jackals in Georgia, which could be related to the late expansion of jackals into the Caucasus region (not before the Bronze Age). Clustering using a Bayesian approach based on the microsatellite markers suggests that the vast majority of both jackals and wolves sampled in western Georgia descend from recent migrants from the east of the country. The expansion of the two species may be related to the conservation efforts in the latest decades or/and climate change that explains the appearance of jackals in the mountain regions of Georgia, as well as in northern Europe.

Effect of time of therapy with wild-type lytic bacteriophages on the reduction of Salmonella Enteritidis in broiler chickens

Salmonella Enteritidis remains a leading cause of human foodborne disease, mostly associated with the consumption of contaminated poultry products. To more strategically implement a phage therapy scheme for S. Enteritidis control in broilers, a cocktail containing three wild-type lytic bacteriophages (LBs) previously isolated from chickens was evaluated shortly and later after a challenge. Genomic characterization, lytic spectrum and in vitro efficacy were determined for each studied LB. In independent trials, broilers challenged with S. Enteritidis on day of hatch received phage therapy from 6 to 10 days of age (early treatment), and from 31 to 35 days of age (later treatment). S. Enteritidis analyses were performed before treatment and at 1, 4, 7 and 10 days post-treatment (dpt) in both trials. Partial DNA sequence analysis of each LB revealed close similarity to the Ackermannviridae family. LBs lysed different Salmonella enterica serovars, while other tested bacteria were refractory. An in-vitro reduction of 1.49, 0.65 and 0.58 log10 CFU/mL in S. Enteritidis number was obtained after co-incubation for 3 h with each LB. Both in vivo trials showed a significant reduction in the average number of intestinal S. Enteritidis calculated after phage therapy compared with controls. However, the highest efficiency was found in the later therapy, which resulted in a reduction of 1.08 log10 CFU/g in the average from 4 to 10 dpt, showing potential for future use as a pre-harvest strategy to reduce the S. Enteritidis intestinal colonization in broilers on farms.

Complete mitochondrial DNA sequence of the endemic Japanese pond turtle Mauremys japonica (Reptilia: Testudines)

The complete mitochondrial genome of the Japanese pond turtle Mauremys japonica from Japan was analyzed using next-generation sequencing. The mitochondrial genome of M. japonica was identified as a 16,443 bp circular molecule containing 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes, along with 1 A + T-rich control region; the average AT content was 62.82%. One extra base was present at the 174th position in the ND3 gene. The phylogenetic analyses of the complete mitochondrial DNA genes strongly supported the result obtained from the phylogenetic analysis of partial DNA sequences, grouping the monophyletic species within the genus Mauremys.

Molecular confirmation of the occurrence of Elysia cf. tomentosa (Mollusca: Heterobranchia) in the Persian Gulf

The molluscan fauna of the Persian Gulf has recently been relatively well documented, yet there are few records of heterobranch sea slugs (opisthobranchs) from the Arabian parts and no report from the Iranian waters. Here we report for the first time the occurrence of one of these molluscs in the northern Persian Gulf (Bandar Abbas, Iran). Sacoglossan specimens were collected in association with the seaweed, Caulerpa sertularioides. Since morphological attributes were not adequately reliable for species identification, molecular approaches were carried out. Maximum-likelihood and Bayesian Inference analysis of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) locus were used for DNA barcoding of large-bodied specimens of Elysia. All Persian Gulf specimens were genetically confirmed as Elysia cf. tomentosa sp. 5, one of at least five morphologically similar but genetically distinct species in the taxonomically challenging and unresolved E. tomentosa complex. This species has previously been recorded only from Australia and Thailand and our finding adds another distant point to the geographic distribution of this species.

Deep-sea Entoprocta from the Sea of Okhotsk and the adjacent open Pacific abyssal area: New species and new taxa of host animals

The Entoprocta fauna still remains almost unexplored: many geographical areas have not yet been studied for their diversity, and almost nothing is known about deep-sea species. We described four new species of solitary Entoprocta from the family Loxosomatidae. The material was collected within a depth range between 3206 m and 3580 m during the German-Russian deep-sea expedition SokhoBio aboard R/V Akademik Lavrentyev in July - August 2015 in the Kuril Basin of the Sea of Okhotsk, Bussol Strait, and the adjacent open Pacific abyssal area. We used methods of light and scanning electronic microscopy and also laser confocal microscopy for morphological studies. Maximum likelihood and Bayesian analyses of partial DNA sequences of 18S and 28S rRNA genes were used to reconstruct phylogenetic trees. Loxosomella aeropsis sp. nov. was found on spines of the sea urchin Aeropsis fulva; Loxosomella cyatiformis sp. nov. was found on shells of the propeamussiid bivalve Catillopecten squamiformis; Loxosomella malakhovi sp. nov. was found living as an epibiont of the nephtyid polychaete Aglaophamus sp., and Loxosomella sextentaculata sp. nov., as an epibiont of the spionid polychaete Laonice sp. Echinoidea and Bivalvia have been first recorded as hosts of loxosomatid entoprocts in their symbiotic associations. A molecular phylogenetic analysis of partial 18S rRNA and 28S rRNA of Loxosomella aeropsis sp. nov., L. cyatiformis sp. nov., and L. malakhovi sp. nov. show that these species nest in the clade of solitary entoprocts among other Loxosomella species. Musculature of investigated species, Loxosomella aeropsis sp. nov and L. malakhovi sp. nov., is typical for loxosomatids and includes the longitudinal muscles of a stalk, the musculature of the digestive tract, the atrial muscular ring, and tentacle muscles. Each species has species-specific muscles so details of myoanatomy can be used for identification of species in addition to other morphological features.

Bioactivities of cry gene positive Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) strains on Ephestia kuehniella Zeller, 1879 and Plodia interpunctella (Hübner, 1813) (Lepidoptera: Pyralidae)

Bacillus thuringiensis is the bacterium most commonly used for biopesticide production due to parasporal crystal formation during its growth cycle. As a consequence of repeated use, B. thuringiensis biopesticides may cause the development of resistance in the pests. Therefore, it is necessary to explore new B. thuringiensis strains with a certain degree of bioactivity. In this study (2012-2013), the bioactivity of native B. thuringiensis strains from the Aegean Region of Turkey were tested against second instar larvae of Ephestia kuehniella and Plodia interpunctella . The bioactivity of 21 B. thuringiensis strains with cry1 , cry2 or cry9 gene was determined as percent mortality according to Abbott’s formula . The highest mortality rates were 42 and 63% in E. kuehniella and P. interpunctella , respectively. These mortality rates were equal to or 1.8 times greater than that of B. thuringiensis subsp. kurstaki . In addition, plasmid profiles of B. thuringiensis strains changed between 5-18 kb . Moreover, SDS-PAGE analysis of the most toxic strains indicated the presence of Cry1 and Cry2 proteins . Two different cry2 gene profiles containing either cry2Aa1 or combination of cry2Aa1 and cry2Ab2 genes were detected by PCR analysis . In addition, partial DNA sequence analysis of cry2A genes indicated phylogenetic differences among the toxic strains and B. thuringiensis subsp. kurstaki . As a result, these B. thuringiensis strains may be used to control both E. kuehniella and P. interpunctella as alternative biopesticides in cases of insect resistance to currently used B. thuringiensis preparations.

New species and new records of Aleurodiscus s.l. (Basidiomycota) in China

Two new species from Hunan Province in central China, Acanthobasidium bambusicola and Aleurodiscus effusus, are described and illustrated. Both species have effused basidiocarps, clamped generative hyphae, acanthophyses, gloeocystidia, and amyloid, echinulate basidiospores. Acanthobasidium weirii is proposed as a new combination. In addition, Aleurodiscus ljubarskii, A. wakefieldiae, and Neoaleurodiscus fujii are reported from mainland China for the first time. Descriptions and illustrations of these three species are included. A preliminary molecular phylogenetic assessment of 11 species of Aleurodiscus sensu lato in China is presented. Phylogenetic relationships were inferred from analyses of partial DNA sequences of the nuclear large subunit ribosomal RNA (nLSU) and a combined dataset of the internal transcribed spacer (ITS) region, nLSU, and the translation elongation factor 1-α gene sequences (tef1-α).

Aeolidia papillosa(Linnaeus, 1761) (Mollusca: Heterobranchia: Nudibranchia), single species or a cryptic species complex? A morphological and molecular study

Aeolidia papillosa (Linnaeus, 1761) is a well-known aeolidiid species that has been reported to have a worldwide distribution in cold–temperate waters, mainly from the northern hemisphere. Molecular tools have recently shown that most cosmopolitan species usually belong to a taxonomic species complex. Here we used integrative taxonomy to test the range of distribution of A. papillosa, and to assess the existence of a putative species complex that has been traditionally included as a single species under the name A. papillosa. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene histone 3, were used to infer phylogenetic trees. Automatic Barcode Gap Discovery (ABGD) species delimitation analyses and morphological study complemented the phylogenetic approach. Our results show that A. papillosa is a cosmopolitan and an amphi-Atlantic species, being distributed in the eastern and western Atlantic as well as in the eastern Pacific; however, some specimens from the UK and the Netherlands, together with specimens from Portugal, Galicia, and France, as well as the Californian and Oregon populations, emerge as two pseudocryptic species described herein: Aeolidia filomenae sp. nov. and Aeolidia loui sp. nov., respectively. Finally, the specimens from Chilean coasts, previously attributed to A. papillosa, belong to a different species, Aeolidia campbellii (Cunningham, 1871), that is a senior synonym of Aeolidia serotinaBergh, 1873.

First record of the Aeolid Anteaeolidiella fijensis (Nudibranchia, Aeolidiidae) from India

The aeolid Anteaeolidiella fijensis Carmona, Bhave, Salunkhe, Pola, Gosliner and Cervera, 2014 is reported for the first time from India. Differences in the external colouration compared with the original description hampered its identification, and therefore, a molecular approach was needed. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes, and the nuclear gene histone-3 were used to infer phylogenetic trees. ABGD species delimitation analyses and morphological study complemented this contribution. This is the first record of Anteaeolidiella fijensis outside the type locality and extends its distribution range remarkably. Here, we also complete the original description with new data regarding the external colouration and internal anatomy. Finally, the instraspecific variation may be related with an ontogenetic process or be a populationally distinctive feature.

Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis′ economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis′ origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3′-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.)

This research has investigated the effect of two genotypes of maize (Zea mays L.) on the number and diversity of diazotrophic bacteria under different N-fertilization rates. Cultivars NK940 and PAU871 were grown in soil:sand with 0, 40, and 80 kg N ha−1 added as NH4NO3 under controlled conditions for 60 days. The number of diazotrophic bacteria of rhizosphere (S), disinfected roots (R), and stems (T) was determined by most probable number (MPN) method. Diazotrophic bacteria were isolated by N-free media, confirmed by amplification of nifH gene fragments, characterized by amplified 16S rDNA restriction analysis (ARDRA) and (GTG)5 fingerprinting, and identified by partial 16S ribosomal DNA (rDNA) sequence analysis. The diversity of the diazotrophic community from S and R was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis considering the nifH gene. Maize genotype had a marked effect on the number and diversity of endophytic communities, the NK940 community being more abundant and diverse than that of PAU871. Additionally, N-fertilization increased the number and diversity of diazotrophs in endophytic communities but not in rhizosphere samples. One hundred and six diazotrophs were isolated from S, R, and T samples. Pseudomonas and Enterobacter were the dominant and ubiquitous genera isolated and detected by culture-independent method. T-RFLP showed that the N-fixing populations of the rhizosphere of both cultivars were more diverse than those of inside roots. Principal component analysis (PCA) separated the samples by cultivar and demonstrated a more marked effect of N-fertilization on the NK940 diazotrophic community than on PAU871. These results support the hypothesis that plant genotype and fertilization conditions should be taken into account when searching for N-fixer inocula.

Evidence for a specific host-endosymbiont relationship between 'Rickettsia sp. genotype RF2125' and Ctenocephalides felis orientis infesting dogs in India.

BackgroundFleas of the genus Ctenocephalides serve as vectors for a number of rickettsial zoonoses, including Rickettsia felis. There are currently no published reports of the presence and distribution of R. felis in India, however, the ubiquitous distribution of its vector Ctenocephalides felis, makes it possible that the pathogen is endemic to the region. This study investigates the occurrence of Rickettsia spp. infection in various subspecies of C. felis infesting dogs from urban areas of Mumbai, Delhi and Rajasthan in India.MethodsIndividual fleas collected off 77 stray dogs from Mumbai, Delhi and Rajasthan were screened for Rickettsia spp. by a conventional PCR targeting the ompB gene. Further genetic characterisation of Rickettsia-positive fleas was carried out using nested PCR and phylogenetic analysis of partial DNA sequences of the gltA and ompA genes. Ctenocephalides spp. were morphologically and genetically identified by PCR targeting a fragment of cox1 gene.ResultsOverall, 56/77 fleas (72.7%), including 22/24 (91.7%) from Delhi, 32/44 (72.7%) from Mumbai and 2/9 (22.2%) from Rajasthan were positive for Rickettsia DNA at the ompB gene. Sequences of gltA fragments confirmed the amplification of Rickettsia sp. genotype RF2125. The ompA gene of Rickettsia sp. genotype RF2125 was characterised for the first time and shown 96% identical to R. felis. Three species of Ctenocephalides were identified, with the Ctenocephalides felis orientis being the dominant flea species (69/77; 89.6%) in India, followed by Ctenocephalides felis felis (8/77; 10.4%).ConclusionsHigh occurrence of Rickettsia sp. genotype RF2125 in C. felis orientis and the absence of R. felis suggests a specific vector-endosymbiont adaptation and coevolution of the Rickettsia felis-like sp. within subspecies of C. felis.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Uganda.

In June 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the booting stage in the Doho rice irrigation scheme, Butaleja district, and at the tillering stage in Nambale, Iganga district and Magada, Namutumba district of Uganda. In areas surveyed, disease incidence was about 80, 40, and 30% in Doho, Nambale, and Magada, respectively. Outside the irrigation schemes, it was lower but widespread. Affected leaves showed typical BLS symptoms, such as water-soaked lesions, translucent stripes, and yellow-brown to black streaks, sometimes with visible exudates at the leaf surfaces. To check for the presence of the bacteria, symptomatic leaves were ground in sterile water and the suspension obtained was subjected to a multiplex PCR assay for X. oryzae pathovars, leading to the three diagnostic DNA fragments for X. oryzae pv. oryzicola (3). In parallel, bacterial strains were isolated from surface-sterilized symptomatic leaves. To this end, rice leaves were ground in sterile distilled water and serial dilutions of the cell suspensions were plated on semi-selective PSA medium (4). Each of the three samples yielded yellow, mucoid Xanthomonas-like colonies that resembled the positive control strain MAI10 (1). These isolates were named Ug_1, Ug_10, and Ug_14, which originated from Doho, Magada, and Nambale, respectively. Multiplex PCR on the pure cultures strongly supported that these isolates corresponded to X. oryzae pv. oryzicola. Two isolates, Ug_1 and Ug_14, were further subjected to partial DNA sequence analysis of the gyrB gene upon PCR amplification using the primers XgyrB1F and XgyrB1R (5). The 467-bp DNA sequence was identical to the gyrB sequences from the X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), and MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of strain Ug_1 was submitted to GenBank (KJ921786). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cultivars Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. For this purpose, bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water at a concentration of 1 × 108 CFU/ml. Bacterial suspensions were sprayed on leaves of rice seedlings. Four seedlings per accession and isolate were inoculated. Fifteen days after incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), inoculated leaves exhibited typical water-soaked lesions with yellow exudates that were similar to the symptoms seen in the fields. Re-isolation of the bacteria from the diseased leaves yielded colonies with the typical morphology of Xanthomonas. Multiplex PCR and sequence analysis of portions of the gyrB gene confirmed that these isolates are X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. One of the three isolates, Ug_1, has been deposited in the Collection Française de Bactéries Phytopathogènes (CFBP) as strain CFBP 8171 ( http://www.angers-nantes.inra.fr/cfbp/ ). Further surveys and strain collections in East and Central Africa will help assess the geographic distribution and importance of BLS.

Detection of Tomato yellow leaf curl virus in imported tomato fruit in northern Europe

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper‐based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full‐length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV‐IL for the Estonian isolate and at 97% to TYLCV‐Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.

Transfer of Kaempferia candida to Curcuma (Zingiberaceae) based on morphological and molecular data

Results of morphological studies supported by molecular analyses of chloroplast psbA-trnH and partial petA-psbJ DNA sequences indicate that Kaempferia candida cannot be retained in this genus. The species is here formally transferred to Curcuma and the name is typified. Notes on the species and colour plates are given.

Amphi-Atlantic distributions and cryptic species in Sacoglossan sea slugs

Nearly 13% of opisthobranch gastropods in the Atlantic Ocean are regarded as amphi-Atlantic (i.e. species occurring on both eastern and western coastlines of the Atlantic realm). This assumption has been broadly based on morpho-anatomical similarities and has rarely been tested within a molecular phylogenetic framework. Among opisthobranchs, the order Sacoglossa is renowned for its difficult and confusing species taxonomy, particularly in some genera like Elysia, where several cases of cryptic speciation have been documented. Moreover, the width of the Atlantic realm poses a serious challenge to the idea of trans-Atlantic dispersal, particularly in nonplanktotrophic species. The species Bosellia mimetica, Elysia timida and Thuridilla picta are three sacoglossan opisthobranchs with amphiAtlantic status, but doubts have been raised about the conspecificity of eastern and western populations. Here we used methods to test molecular phylogenetic whether the Atlantic Ocean constitutes a barrier for dispersal and hence whether eastern and western populations belong to the same species. We used the criteria of divergence and reciprocal monophyly, supported by concordant genealogically independent genetic markers, to define species. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes and nuclear gene Histone-3 (H3) were used to produce phylogenetic trees. We conclude that the eastern Atlantic species B. mimetica is not amphi-Atlantic, although the H3 gene did not recover reciprocal monophyly between western and eastern populations, perhaps in a result of incomplete lineage-sorting. Elysia timida is the only species showing an amphi-Atlantic distribution, but possibly due to recent introduction in the tropical western Atlantic. Thuridilla picta is restricted to the western Atlantic and therefore not amphi-Atlantic. Populations of ‘T. picta’ inhabiting the Macaronesian archipelagos of Azores and Madeira are a colour form of the eastern Atlantic T. hopei. Our results highlight the difficulty surrounding systematics of Sacoglossa sea slugs and provide further evidence that cryptic speciation is common in the group.