A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding

Despite their relatively complex morphologies, species in the genus Alaria Greville are notoriously difficult to identify with certainty. Morphological characters, often influenced by environmental factors, make individuals in similar habitats artificially appear related. Species identification would, therefore, benefit greatly from the application of molecular tools. We applied DNA barcoding, using the 5' end of the cytochrome c oxidase I (coxI-5') gene from the mitochondrial genome, to define species limits and relationships in northeast Pacific populations of Alaria. This emerging technique is being employed to catalogue species diversity worldwide, particularly among animals, and it has been shown to be sensitive enough to discriminate between closely related species. However, the utility of this marker for identifying or categorizing the majority of life remains unclear. We compared the resolution obtained with this marker to two other molecular systems commonly used in algal research: the nuclear internal transcribed spacer (ITS) of the ribosomal cistron, and the plastid Rubisco operon spacer (rbcSp). In agreement with previous results, Alaria fistulosa Postels & Ruprecht, with its distinct morphological, ecological and molecular features, stands apart from the other species in the genus and we establish Druehlia gen. nov. to accommodate it. For the remaining isolates, distinct mitochondrial haplotypes resolved with the barcode data indicate a period of genetic isolation for at least three incipient species in the northeast Pacific, whereas unexpected levels and patterns of ITS variation, as well as the extreme morphological plasticity found among these isolates, have most probably resulted from a recent collapse in species barriers. The cloning of ITS amplicons revealed multiple ITS copies in several individuals, further supporting this hypothesis.     

Skull morphology of bottlenose dolphins from different ocean populations with emphasis on South America

The bottlenose dolphin, genus Tursiops, is cosmopolitan occurring in tropical and temperate regions, with morphological variation between and within different oceans. Since the genus' taxonomy has been under discussion for a long time, this work aimed at analyzing the cranial variability of T. truncatus from different regions of the world. Geometric Morphometrics analyses were performed in 201 skulls of adult specimens, on dorsal, ventral, and lateral views, from the Eastern North Pacific, Eastern North Atlantic, Eastern South Atlantic, and Western South Atlantic oceans. The results indicate differences between individuals that inhabit the Atlantic and Pacific oceans. Within the Atlantic Ocean, there is an evident longitudinal differentiation of specimens from the eastern and western regions. A latitudinal separation was also observed, considering specimens from the North and South Atlantic Ocean. In the Western South Atlantic statistical differences were found between two morphological groups, identified as T. gephyreus (sensu Lahille, 1908) and T. truncatus, and the cross-validation presented 98% as minimum confidence for correct classification of these two groups. The present study provides strong morphological support to consider these two lineages as separate species.     

A mtDNA-based phylogeny of the brown algal genus Fucus (Heterokontophyta; Phaeophyta)

Species of Fucus are among the dominant seaweeds along Northern Hemisphere shores, but taxonomic designations often are confounded by significant intraspecific morphological variability. We analyzed intra- and inter-specific phylogenetic relationships within the genus (275 individuals representing 16 taxa) using two regions of the mitochondrion: a variable intergenic spacer and a conserved portion of the 23S subunit. Bayesian ML and MP analyses verified a shallow phylogeny with two major lineages (previously reported) and resolved some intra-lineage relationships. Significant species-level paraphyly/polyphyly was observed within lineages 1A and 2. Despite higher species richness in the North Atlantic, a North Pacific origin of the genus is supported by a gradient of decreasing haplotype and nucleotide diversities in F. distichus from the North Pacific to the East Atlantic.     

Reexamination of the Species Assignment of Diacavolinia Pteropods Using DNA Barcoding

Thecosome pteropods (Mollusca, Gastropoda) are an ecologically important, diverse, and ubiquitous group of holoplanktonic animals that are the focus of intense research interest due to their external aragonite shell and vulnerability to ocean acidification. Characterizing the response of these animals to low pH and other environmental stressors has been hampered by continued uncertainty in their taxonomic identification. An example of this confusion in species assignment is found in the genus Diacavolinia. All members of this genus were originally indentified as a single species, Cavolinia longirostris, but over the past fifty years the taxonomy has been revisited multiple times; currently the genus comprises 22 different species. This study examines five species of Diacavolinia, including four sampled in the Northeast Atlantic (78 individuals) and one from the Eastern tropical North Pacific (15 individuals). Diacavolina were identified to species based on morphological characteristics according to the current taxonomy, photographed, and then used to determine the sequence of the “DNA barcoding” region of the cytochrome c oxidase subunit I (COI). Specimens from the Atlantic, despite distinct differences in shell morphology, showed polyphyly and a genetic divergence of <3% (K2P distance) whereas the Pacific and Atlantic samples were more distant (∼19%). Comparisons of Diacavolinia spp. with other Cavolinia spp. reveal larger distances (∼24%). These results indicate that specimens from the Atlantic comprise a single monophyletic species and suggest possible species-level divergence between Atlantic and Pacific populations. The findings support the maintenance of Diacavolinia as a separate genus, yet emphasize the inadequacy of our current taxonomic understanding of pteropods. They highlight the need for accurate species identifications to support estimates of biodiversity, range extent and natural exposure of these planktonic calcifiers to environmental variability; furthermore, the apparent variation of the pteropods shell may have implications for our understanding of the species’ sensitivity to ocean acidification.     

A new molecular phylogenetic hypothesis for the evolution of freshwater eels

Phylogenetic analysis of a segment of the mitochondrial 16S rDNA of eight Anguilla species from the Indo-Pacific region and from the North Atlantic revealed that the genus Anguilla appears to be surprisingly young, based upon the small observed maximum genetic distance of 4.8% and the high degree of morphological similarity among the species. The placement of A. marmorata as the most ancestral lineage suggests that the genus is likely to have originated in the Indo-Malayian region, from which it quickly spread. Two Pacific species, A. obscura and A. japonica, branched next. A. japonica was placed as sister group to all remaining species, which formed three clades: the first comprising A. australis, the second A. reinhardti and A. mossambica, and the third A. anguilla and A. rostrata. All analyzed specimens of A. rostrata originating from southern New Jersey to Nova Scotia had identical mitotypes, while five mitochondrial genotypes were found in Europe differing by zero to two substitutions. The two Atlantic eel species are very closely related; all surveyed specimens of A. anguilla differ by three to five substitutions from their American allies, corroborating the existence of two distinct biological species. This was also confirmed by restriction analysis of a 350-bp segment of the cytochrome b, in which American specimens were distinct in sharing a single diagnostic restriction site of HinfI. Our results suggest little to no gene flow between the two nominal Atlantic eel species.     

THE BIOGEOGRAPHIC ORIGIN OF ARCTIC ENDEMIC SEAWEEDS: A THERMOGEOGRAPHIC VIEW1

The Arctic is geologically and biogeographically young, and the origin of its seaweed flora has been widely debated. The Arctic littoral biogeographic region dates from the latest Tertiary and Pleistocene. Following the opening of Bering Strait, about 3.5 mya, the “Great Trans‐Arctic Biotic Interchange” populated the Arctic with a fauna strongly dominated by species of North Pacific origin. The Thermogeographic Model (TM) demonstrates why climate and geography continued to support this pattern in the Pleistocene. Thus, Arctic and Atlantic subarctic species of seaweeds are likely to be evolutionarily “based” in the North Pacific, subarctic species are likely to be widespread in the warmer Arctic, and species of Atlantic Boreal or warmer origin are unlikely in the Arctic and Subarctic. Although Arctic seaweeds have been thought to have a greater affinity with the North Atlantic, we have reanalyzed the Arctic endemic algal flora, using the Thermogeographic Model and evolutionary trees based on molecular data, to demonstrate otherwise. There are 35 congeneric species of the six, abundant Arctic Rhodophyta that we treat in this paper; 32 of these species (91%) occur in the North Pacific, two species (6%) occur in the Boreal or warmer Atlantic Ocean, and a single species is panoceanic, but restricted to the Subarctic. Laminaria solidungula J. Agardh, a kelp Arctic “endemic” species, has 18 sister species. While only eleven (61%) occur in the North Pacific, this rapidly dispersing and evolving genus is a terminal member of a diverse family and order (Laminariales) widely accepted to have evolved in the North Pacific. Thus, both the physical/time‐based TM and the dominant biogeographic pattern of relatives of Arctic macrophytes suggest strong compliance with the evidence of zoology, geology, and paleoclimatology that the Arctic marine flora is largely of Pacific origin.     

Species diversity of the deep‐water gulper sharks (Squaliformes: Centrophoridae: Centrophorus) in North Atlantic waters – current status and taxonomic issues

The gulper sharks (genus Centrophorus) are a group of deep‐water benthopelagic sharks with a worldwide distribution. The alpha taxonomy of the group has historically been problematic and the number of species included in the genus has varied considerably over the years and is still under debate. Gulper sharks are routinely caught in mid‐ and deep‐water fisheries worldwide and some have shown a considerable decline in abundance in the last few decades. Clear and consistent species discrimination of Centrophorus is essential for an efficient and sustainable management of these fisheries resources. Our study used molecular cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene sequences and morphometric data to re‐evaluate the diversity of Centrophorus in North Atlantic waters, including the Gulf of Mexico, the Caribbean, and the Mediterranean Seas. Molecular data separated North Atlantic Centrophorus into five well‐supported groups whereas morphometric data separated these same five groups and suggested three additional groups for which no molecular data were available. Four of the five groups identified in the North Atlantic also occur in the Indian and/or Pacific Oceans, thus extending the reported range of some species considerably. A species identification key for North Atlantic Centrophorus is provided based on our findings. © 2014 The Linnean Society of London     

Phylogenetics of Cancer crabs (Crustacea: Decapoda: Brachyura).

We used morphological, mitochondrial DNA sequence, paleontological, and biogeographical information to examine the evolutionary history of crabs of the genus Cancer. Phylogenies inferred from adult morphology and DNA sequence of the cytochrome oxidase I (COI) gene were each well resolved and well supported, but differed substantially in topology. Four lines of evidence suggested that the COI data set accurately reflected Cancer phylogeny: (1) in the phylogeny inferred from morphological data, each Atlantic species was sister taxon to an ecologically similar Pacific species, suggesting convergence in morphology; (2) a single trans-Arctic dispersal event, as indicated by the phylogeny inferred from COI, is more parsimonious than two such dispersal events, as inferred from morphology; (3) test and application of a maximum likelihood molecular clock to the COI data yielded estimates of origin and speciation times that fit well with the fossil record; and (4) the tree inferred from the combined COI and morphology data was closely similar to the trees inferred from COI, although notably less well supported by the bootstrap. The phylogeny inferred from maximum likelihood analysis of COI suggested that Cancer originated in the North Pacific in the early Miocene, that the Atlantic species arose from a North Pacific ancestor, and that Cancer crabs invaded the Atlantic from the North Pacific 6-12 mya. This inferred invasion time is notably prior to most estimates of the date of submergence of the Bering Strait and the trans-Arctic interchange, but it agrees with fossil evidence placing at least one Cancer species in the Atlantic about 8 mya.     

Comparative characteristics of North Pacific and North Atlantic specimens of a rare bathypelagic fish <Emphasis Type="Italic">Thalassobathia pelagica</Emphasis> (Teleostei: Bythitidae) with discussion of its distribution

A detailed morphological comparison is made of the North Pacific specimen of Thalassobathia pelagica with fish from the North Atlantic (the type locality). The Pacific specimen is recognized as conspecific with Atlantic ones. Insignificant differences are considered as age or individual variation. The structure of the seismosensory system of T. pelagica and morphology of this species are described in more detail. The probable distribution of T. pelagica is discussed. A wide tropical area of this species is assumed and a possibility of its presence in all three oceans. The captures of T. pelagica in high latitudes may depend on its transportation with warm currents.     

Morphology,microhabitats and geographical variation of Kuhnia spp. (Monogenea: Polyopisthocotylea)

Kuhnia scombri and K. sprostonae are the only species of the genus recognized as valid. Hosts are scombrid fishes and possibly the genus Scomber only. Examination of many populations from localities in the Atlantic and Pacific showed much geographical variation. Variation is greatest in K. sprostonae, with a distinctly different population on S. scombrus in the North Atlantic. There are intermediate stages in all characters between different populations of both species (except possibly K. sprostonae in the North Atlantic). Some populations within each species have different microhabitats on the gills and pseudobranchs. Differences between parasite populations correspond to some degree to those of host populations, but at least some of the differences are non-adaptive.     

Molecular evidence of new variant Brucella in North Pacific common minke whales

Brucella, a causative agent of brucellosis, has been isolated recently from a variety of marine mammals. The molecular analysis of marine mammalian Brucella strains, without manifest pathology of brucellosis in the eastern North Atlantic, showed that they are distinct from terrestrial Brucella species. Previously, we reported abnormal gonads in common minke whales (Balaenoptera acutorostrata) in the western North Pacific and suggested the presence of Brucella infection in the whales in pathology and serology studies. In the present study, using polymerase chain reaction (PCR), Brucella was detected in granular testes of the whales showing caseation or calcification. The insertion of an IS711 transposable element specific for marine mammal isolates as well as a seal isolate-specific DNA fragment were also found. Molecular characterization of Brucella based on sequence analysis of the PCR products amplified from the outer membrane protein (omp) 2 gene showed that the Brucella from North Pacific common minke whales was different from terrestrial and North Atlantic marine mammal Brucella strains. The North Pacific Brucella showed the highest similarity to North Atlantic seal strains among the known Brucella strains.     

A review of the pelagic ophidioid fish genus Brotulataenia with descriptions of two new species

An osteological study and a taxonomic diagnosis are presented for the mesopelagic, oviparous ophidioid fish genus Brotulataenia. Its relationships are probably with a large group of benthic deep-sea oviparous genera. Brotulataenia has been known previously from three North Atlantic specimens assigned to two species. The present study is based on 33 specimens from the North and South Atlantic and Pacific Oceans and the Indian Ocean, and placed in four species, two of which are described as new. Two species are excessively elongate and are interpreted as being derived forms; they are lighter in color than are the two short-bodied species and may live higher in the water column.     

Intraspecific sterility barrier confirms that introduction of Sphaerotrichia divaricata (Phaeophyceae,Chordariales) into the Mediterranean was from Japan

Crossing studies revealed an intraspecific sterility barrier on the level of zygote formation between Japanese Sphaerotrichia divaricata and isolates of the same species from the Northeast Pacific and the North Atlantic. Because no consistent morphological differences exist between sporophytes from Japan and other areas, we propose not to distinguish the intersterile populations as different species. Japanese Sphaerotrichia and isolates from a recently detected population in the Étang de Thau, French Mediterranean coast, are interfertile. The crossing studies support the assumption that this Mediterranean population is a recent introduction from Japan.     

24 Diversity of macroalgal epiphytes on Thalassia and Syringodium in tampa bay, florida

Alaria is a common kelp genus generally found in the lower intertidal and shallow subtidal regions of rocky shores in the cold waters of the northern Hemisphere. About 16 species are currently recognized worldwide and, of these, A. fistulosa is distinguished by having hollow midrib and large blades with 10–30 m in length and 30–90 cm in width. It is the only canopy-producing kelp in the northwest Pacific, where it is restricted to the waters of north Hokkaido, Kamchatka, Aleutian Islands, and Alaska. In order to know the phylogenetic position of A. fistulosa , sequences of nr DNA ITS and plastid rbc L including spacer and psaA regions were determined in A. fistulosa and compared with homologous positions of newly sequenced putative relatives and with published sequences of other kelp species. Combined data of ITS and Rubisco spacer show that A. fistulosa was more related to the clade of Lessoniopsis and Pterygophora than to the clade of other species of Alaria , which is supported by the rbc L and psaA sequence data. The topologies from nuclear and plastid DNA sequences lead to phylogenetic independence of A. fistulosa , which is clearly different from the genus Alaria .     

Evolutionary diversification of banded tube-dwelling anemones (Cnidaria; Ceriantharia; Isarachnanthus) in the Atlantic Ocean

The use of molecular data for species delimitation in Anthozoa is still a very delicate issue. This is probably due to the low genetic variation found among the molecular markers (primarily mitochondrial) commonly used for Anthozoa. Ceriantharia is an anthozoan group that has not been tested for genetic divergence at the species level. Recently, all three Atlantic species described for the genus Isarachnanthus of Atlantic Ocean, were deemed synonyms based on morphological simmilarities of only one species: Isarachnanthus maderensis. Here, we aimed to verify whether genetic relationships (using COI, 16S, ITS1 and ITS2 molecular markers) confirmed morphological affinities among members of Isarachnanthus from different regions across the Atlantic Ocean. Results from four DNA markers were completely congruent and revealed that two different species exist in the Atlantic Ocean. The low identification success and substantial overlap between intra and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding, which is not true for Ceriantharia. In addition, genetic divergence within and between Ceriantharia species is more similar to that found in Medusozoa (Hydrozoa and Scyphozoa) than Anthozoa and Porifera that have divergence rates similar to typical metazoans. The two genetic species could also be separated based on micromorphological characteristics of their cnidomes. Using a specimen of Isarachnanthus bandanensis from Pacific Ocean as an outgroup, it was possible to estimate the minimum date of divergence between the clades. The cladogenesis event that formed the species of the Atlantic Ocean is estimated to have occured around 8.5 million years ago (Miocene) and several possible speciation scenarios are discussed.     

Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants

A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44.113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola.     

Phylogeny and biogeography of Muusoctopus (Cephalopoda: Enteroctopodidae)

Deep‐sea octopuses of the genus Muusoctopus are thought to have originated in the Pacific Northern Hemisphere and then diversified throughout the Pacific and into the rest of the World Ocean. However, this hypothesis was inferred only from molecular divergence times. Here, the ancestral distribution and dispersal routes are estimated by Bayesian analysis based on a new phylogeny including 38 specimens from the south‐eastern Pacific Ocean. Morphological data and molecular sequences of three mitochondrial genes (16S rRNA, COI and COIII) are presented. The morphological data confirm that specimens newly acquired from off the coast of Chile comprise two species: Muusoctopus longibrachus and the poorly described species, Muusoctopus eicomar. The latter is here redescribed and is clearly distinguished from M. longibrachus and other closely related species in the region. A gene tree was built using Bayesian analysis to infer the phylogenetic position of these species within the species group, revealing that a large genetic distance separates the two sympatric Chilean species. M. longibrachus is confirmed as the sister species of Muusooctopus eureka from the Falkland Islands; while M. eicomar is a sister species of Muusoctopus yaquinae from the North Pacific, most closely related to the amphi‐Atlantic species Muusoctopus januarii. Molecular divergence times and ancestral distribution analyses suggest that genus Muusoctopus may have originated in the North Atlantic: one lineage dispersed directly southward to the Magellan region and another dispersed southward along the Eastern Pacific to the Southern Ocean and Antarctica. The Muusoctopus species in the Southern Hemisphere have different phylogenetic origins and represent independent invasions of this region.     

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH ATLANTIC1

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5–13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte. The second group of samples, from Nova Scotia and Northern Ireland, provisionally referred to as Gymnogongrus sp., showed little morphological variation. The life history in both areas consists of apomictically reproducing diploid female gametophytes and diploid crustose bisporophytes and tetrasporophytes. Rubisco spacer sequences of the samples were identical, and the plasmid previously described in the Nova Scotian samples was also present in the Northern Ireland population. This species is widely distributed in the western Atlantic, from Newfoundland to Massachusetts. In Europe, gametophytes are known only at one site, but crusts are distributed from Denmark, Scotland (and probably Norway) to France. It is very likely that this species was introduced from one side of the North Atlantic to the other by shipping during the early nineteenth century. Several morphological features are unusual within the genus but are shared with G. leptophyllus J. Agardh from the eastern Pacific Ocean, and further work is necessary to determine whether Gymnogongrus sp. and G. leptophyllus are conspecific.     

Low worldwide genetic diversity in the basking shark (Cetorhinus maximus)

The basking shark (Cetorhinus maximus) is found in temperate waters throughout the world's oceans, and has been subjected to extensive exploitation in some regions. However, little is known about its current abundance and genetic status. Here, we investigate the diversity of the mitochondrial DNA control region among samples from the western North Atlantic, eastern North Atlantic, Mediterranean Sea, Indian Ocean and western Pacific. We find just six haplotypes defined by five variable sites, a comparatively low genetic diversity of pi=0.0013 and no significant differentiation between ocean basins. We provide evidence for a bottleneck event within the Holocene, estimate an effective population size (Ne) that is low for a globally distributed species, and discuss the implications.