High Cryptic Diversity across the Global Range of the Migratory Planktonic Copepods Pleuromamma piseki and P. gracilis

Although holoplankton are ocean drifters and exhibit high dispersal potential, a number of studies on single species are finding highly divergent genetic clades. These cryptic species complexes are important to discover and describe, as identification of common marine species is fundamental to understanding ecosystem dynamics. Here we investigate the global diversity within Pleuromamma piseki and P. gracilis, two dominant members of the migratory zooplankton assemblage in subtropical and tropical waters worldwide. Using DNA sequence data from the mitochondrial gene cytochrome c oxidase subunit II (mtCOII) from 522 specimens collected across the Pacific, Atlantic and Indian Oceans, we discover twelve well-resolved genetically distinct clades in this species complex (Bayesian posterior probabilities >0.7; 6.3–17% genetic divergence between clades). The morphologically described species P. piseki and P. gracilis did not form monophyletic groups, rather they were distributed throughout the phylogeny and sometimes co-occurred within well-resolved clades: this result suggests that morphological characters currently used for taxonomic identification of P. gracilis and P. piseki may be inaccurate as indicators of species’ boundaries. Cryptic clades within the species complex ranged from being common to rare, and from cosmopolitan to highly restricted in distribution across the global ocean. These novel lineages appear to be ecologically divergent, with distinct biogeographic distributions across varied pelagic habitats. We hypothesize that these mtDNA lineages are distinct species and suggest that resolving their systematic status is important, given the ecological significance of the genus Pleuromamma in subtropical-tropical waters worldwide.     

Worldwide genetic differentiation in the common fouling barnacle,Amphibalanus amphitrite

Amphibalanus amphitrite is a common fouling barnacle distributed globally in tropical and subtropical waters. In the present study, the genetic (mitochondrial cytochrome oxidase subunit I) and morphological differentiation in A. amphitrite from 25 localities around the world were investigated. The results revealed three clades within A. amphitrite with a genetic divergence of ~ 4% among clades, whereas there were no diagnostic morphological differences among clades. Clade 1 is widely distributed in both temperate and tropical waters, whereas Clade 3 is currently restricted to the tropical region. The deep divergence among clades suggests historical isolation within A. amphitrite; thus, the present geographical overlaps are possibly a result of the combined effects of rising sea level and human-mediated dispersals. This study highlights the genetic differentiation that exists in a common, widely distributed fouling organism with great dispersal potential; future antifouling research should take into account the choice of lineages.     

Phylogeography of the yucca moth Tegeticula maculata: the role of historical biogeography in reconciling high genetic structure with limited speciation

Tegeticula maculata is one of the most ancient and morphologically variable lineages within the yucca moths, yet has apparently undergone little diversification in comparison with much younger yucca moth lineages that have rapidly diversified. A phylogeographic approach was used to determine the number of independent lineages within T. maculata and to examine whether these patterns corresponded with morphological differences between its subspecies maculata and extranea. Phylogenetic analysis of mitochondrial DNA sequence variation indicated that the two subspecies are in separate clades, but there was also an equally deep split within subspecies maculata. There was no evidence for gene flow among regions and there was considerable substructure within clades. The phylogeographic structure of moth populations among and within subspecies can be explained in part by historical biogeographic boundaries and increasingly patchy postglacial distribution of the exclusive host plant, Hesperoyucca whipplei. Local specialization and co-adaptation would be possible in the absence of apparent gene flow, yet gross morphological divergence is limited to the very old split between the subspecies. Sorting of ancient mitochondrial lineages followed by local genetic differentiation may explain the pattern of high genetic structure with limited speciation.     

Reconstructing the colonisation and diversification history of the endemic freshwater crab (Seychellum alluaudi) in the granitic and volcanic Seychelles Archipelago

The endemic, monotypic freshwater crab species Seychellum alluaudi was used as a template to examine the initial colonisation and evolutionary history among the major islands in the Seychelles Archipelago. Five of the “inner” islands in the Seychelles Archipelago including Mahé, Praslin, Silhouette, La Digue and Frégate were sampled. Two partial mtDNA fragments, 16S rRNA and cytochrome oxidase subunit I (COI) was sequenced for 83 specimens of S. alluaudi. Evolutionary relationships between populations were inferred from the combined mtDNA dataset using maximum parsimony, maximum likelihood and Bayesian inferences. Analyses of molecular variance (AMOVA) were used to examine genetic variation among and within clades. A haplotype network was constructed using TCS while BEAST was employed to date the colonisation and divergence of lineages on the islands. Phylogenetic analyses of the combined mtDNA data set of 1103 base pairs retrieved a monophyletic S. alluaudi group comprised three statistically well-supported monophyletic clades. Clade one was exclusive to Silhouette; clade two included samples from Praslin sister to La Digue, while clade three comprised samples from Mahé sister to Frégate. The haplotype network corresponded to the three clades. Within Mahé, substantial phylogeographic substructure was evident. AMOVA results revealed limited genetic variation within localities with most variation occurring among localities. Divergence time estimations predated the Holocene sea level regressions and indicated a Pliocene/Pleistocene divergence between the three clades evident within S. alluaudi. The monophyly of each clade suggests that transoceanic dispersal is rare. The absence of shared haplotypes between the three clades, coupled with marked sequence divergence values suggests the presence of three allospecies within S. alluaudi.     

Genetic and phylogenetic divergence of feline immunodeficiency virus in the puma (Puma concolor).

Feline immunodeficiency virus (FIV) is a lentivirus which causes an AIDS-like disease in domestic cats (Felis catus). A number of other felid species, including the puma (Puma concolor), carry a virus closely related to domestic cat FIV. Serological testing revealed the presence of antibodies to FIV in 22% of 434 samples from throughout the geographic range of the puma. FIV-Pco pol gene sequences isolated from pumas revealed extensive sequence diversity, greater than has been documented in the domestic cat. The puma sequences formed two highly divergent groups, analogous to the clades which have been defined for domestic cat and lion (Panthera leo) FIV. The puma clade A was made up of samples from Florida and California, whereas clade B consisted of samples from other parts of North America, Central America, and Brazil. The difference between these two groups was as great as that reported among three lion FIV clades. Within puma clades, sequence variation is large, comparable to between-clade differences seen for domestic cat clades, allowing recognition of 15 phylogenetic lineages (subclades) among puma FIV-Pco. Large sequence divergence among isolates, nearly complete species monophyly, and widespread geographic distribution suggest that FIV-Pco has evolved within the puma species for a long period. The sequence data provided evidence for vertical transmission of FIV-Pco from mothers to their kittens, for coinfection of individuals by two different viral strains, and for cross-species transmission of FIV from a domestic cat to a puma. These factors may all be important for understanding the epidemiology and natural history of FIV in the puma.     

Cryptic ecological diversification of a planktonic estuarine copepod, Acartia tonsa

The recent discovery of cryptic species in marine holoplankton, organisms that 'drift' in oceanic currents throughout their life cycle, contrasts with their potential for long-distance passive dispersal and presumably high gene flow. These observations suggest that holoplankton species are adapting to surprisingly small-scale oceanographic features and imply either limited dispersal or strong selection gradients. Acartia tonsa is a widespread and numerically dominant estuarine copepod containing deep mitochondrial lineages within and among populations along the northwestern Atlantic coast. In this study, we intensively investigated A. tonsa populations in Chesapeake Bay with the goals of testing species status for the deep lineages and testing for their association with environmental features over space and time. Phylogenetic analyses of DNA sequences from mitochondrial cytochrome c oxidase I (mtCOI) and the nuclear ribosomal internal transcribed spacer (nITS) resolved two concordant monophyletic clades. Deep divergence between the two clades (13.7% uncorrected sequence divergence for mtCOI and 32.2% for nITS) and genealogical concordance within sympatric populations strongly suggest that the two clades represent reproductively isolated cryptic species. Based on restriction fragment length polymorphisms of mtCOI, representatives from the two clades were found consistently associated with contrasting salinity regimes (oligohaline vs. meso-polyhaline) with an overlap between 2 and 12 PSU in samples from 1995 to 2005. Finding these patterns in one of the best-known estuarine copepods reinforces the conclusion that marine biodiversity is underestimated, not only in terms of species numbers, but also with respect to niche partitioning and the potential importance of ecological divergence in marine holoplankton.     

Cryptic Neogene vicariance and Quaternary dispersal of the red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas

We define the geographical distributions of mitochondrial DNA (mtDNA) lineages embedded within a broadly distributed, arid-dwelling toad, Bufo punctatus. These patterns were evaluated as they relate to hypothesized vicariant events leading to the formation of desert biotas within western North America. We assessed mtDNA sequence variation among 191 samples from 82 sites located throughout much of the species' range. Parsimony-based haplotype networks of major identified lineages were used in nested clade analysis (NCA) to further elucidate and evaluate shallow phylogeographic patterns potentially associated with Quaternary (Pleistocene-Holocene) vicariance and dispersal. Phylogenetic analyses provided strong support for three monophyletic lineages (clades) within B. punctatus. The geographical distributions of the clades showed little overlap and corresponded to the general boundaries of the Peninsular Desert, and two continental desert regions, Eastern (Chihuahuan Desert-Colorado Plateau) and Western (Mojave-Sonoran deserts), geographically separated along the Rocky Mountains and Sierra Madre Occidental. The observed divergence levels and congruence with postulated events in earth history implicate a late Neogene (latest Miocene-early Pliocene) time frame for separation of the major mtDNA lineages. Evaluation of nucleotide and haplotype diversity and interpretations from NCA reveal that populations on the Colorado Plateau resulted from a recent, likely post-Pleistocene, range expansion from the Chihuahuan Desert. Dispersal across historical barriers separating major continental clades appear to be recent, resulting in secondary contacts in at least two areas. Given the observed contact between major clades, we speculated as to why the observed deep phylogeographic structure has not been eroded during the multiple previous interglacials of the Pleistocene.     

Phylogeographic structure in the bogus yucca moth Prodoxus quinquepunctellus (Prodoxidae): comparisons with coexisting pollinator yucca moths

The pollination mutualism between yucca moths and yuccas highlights the potential importance of host plant specificity in insect diversification. Historically, one pollinator moth species, Tegeticula yuccasella, was believed to pollinate most yuccas. Recent phylogenetic studies have revealed that it is a complex of at least 13 distinct species, eight of which are specific to one yucca species. Moths in the closely related genus Prodoxus also specialize on yuccas, but they do not pollinate and their larvae feed on different plant parts. Previous research demonstrated that the geographically widespread Prodoxus quinquepunctellus can rapidly specialize to its host plants and may harbor hidden species diversity. We examined the phylogeographic structure of P. quinquepunctellus across its range to compare patterns of diversification with six coexisting pollinator yucca moth species. Morphometric and mtDNA cytochrome oxidase I sequence data indicated that P. quinquepunctellus as currently described contains two species. There was a deep division between moth populations in the eastern and the western United States, with limited sympatry in central Texas; these clades are considered separate species and are redescribed as P. decipiens and P. quinquepunctellus (sensu stricto), respectively. Sequence data also showed a lesser division within P. quinquepunctellus s.s. between the western populations on the Colorado Plateau and those elsewhere. The divergence among the three emerging lineages corresponded with major biogeographic provinces, whereas AMOVA indicated that host plant specialization has been relatively unimportant in diversification. In comparison, the six pollinator species comprise three lineages, one eastern and two western. A pollinator species endemic to the Colorado Plateau has evolved in both of the western lineages. The east-west division and the separate evolution of two Colorado Plateau pollinator species suggest that similar biogeographic factors have influenced diversification in both Tegeticula and Prodoxus. For the pollinators, however, each lineage has produced a monophagous species, a pattern not seen in P. quinquepunctellus.     

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b, NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogalexanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n=22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia.     

DNA Barcoding in Pencilfishes (Lebiasinidae: Nannostomus) Reveals Cryptic Diversity across the Brazilian Amazon

Nannostomus is comprised of 20 species. Popularly known as pencilfishes the vast majority of these species lives in the flooded forests of the Amazon basin and are popular in the ornamental trade. Among the lebiasinids, it is the only genus to have undergone more than one taxonomic revision. Even so, it still possesses poorly defined species. Here, we report the results of an application of DNA barcoding to the identification of pencilfishes and highlight the deeply divergent clades within four nominal species. We surveyed the sequence variation in the mtDNA cytochrome c oxidase subunit I gene among 110 individuals representing 14 nominal species that were collected from several rivers along the Amazon basin. The mean Kimura-2-parameter distances within species and genus were 2% and 19,0%, respectively. The deep lineage divergences detected in N. digrammus, N. trifasciatus, N. unifasciatus and N. eques suggest the existence of hidden diversity in Nannostomus species. For N. digrammus and N. trifasciatus, in particular, the estimated divergences in some lineages were so high that doubt about their conspecific status is raised.     

Phylogeography and the origins of range disjunctions in a north temperate fish,the pygmy whitefish (Prosopium coulterii), inferred from mitochondrial and nuclear DNA sequence analysis

Aim To investigate the degree of phylogeographical divergence within pygmy whitefish (Prosopium coulterii) and to test hypotheses concerning the origin of disjunct populations within North America. Location North America from western Alaska to Lake Superior. Methods Mitochondrial (ATPase subunit VI) and nuclear (ITS‐1, ITS‐2) DNA sequence variation was assessed across the species’ North American range to test for the existence of distinct phylogeographical groupings of pygmy whitefish associated with known glacial refugia. Coalescent simulations of the mitochondrial DNA (mtDNA) data were used to test hypotheses of population structure. Results This species is composed of two monophyletic mitochondrial clades across its North American range. The two mtDNA clades differed by an average 3.3% nucleotide sequence divergence. These clades were also distinguished by ITS‐2, but the relationships among lineages were not resolved by the ITS‐1 analysis. Coalescent analyses rejected the null hypothesis of the current disjunct distributions being a result of fragmentation of a single widespread ancestral lineage across a variety of effective population sizes and divergence times. Main conclusions The current range disjunctions of pygmy whitefish in North America probably resulted from isolation, genetic divergence, and selective dispersal from at least two major Pleistocene glacial refugia: Beringia and Cascadia. More recent isolation and dispersal from an upper Mississippi refugium is suggested by relationships within one of the clades and by distributional evidence from co‐distributed species. The Beringian and Cascadian refugia have played major roles in the zoogeography of Nearctic temperate aquatics, but the roles of smaller refugia appear more variable among other species.     

Phylogeography of Iberian Discoglossus (Anura: Discoglossidae)

The genus Discoglossus (Anura, Discoglossidae) comprises five morphologically similar species. Molecular analyses have found high values of genetic differentiation among these cryptic taxa and confirmed the existence of two Iberian endemic lineages ( Discoglossus galganoi and D. jeanneae ) and a distinct lineage of uncertain origin representing a third species in NE Spain. In order to analyse the pattern of geographical distribution of haplotype diversity within Iberian Discoglossus and test the hypothesis of an Algerian origin for populations in NE Spain sequence data has been resolved from 35 populations of D. galganoi and D. jeanneae on the Iberian Peninsula and samples from NE Spain and north Africa. I analysed 959 bp corresponding to partial sequences of cytochrome b and nad 4 and found extremely low values of sequence divergence among populations of D. jeanneae . Three clades can be recognized within D. galganoi , however, and a statistically significant association with geography can be explained as a result of past fragmentation. Sequence data strongly support an Algerian origin for populations of Discoglossus from NE Spain. Finally, the taxonomic status of Discoglossus in Morocco is discussed and its elevation to species status as Discoglossus scovazzi Camerano, 1878 is suggested.     

Phylogeography of the leaf beetle Chrysolina virgata in wetlands of Japan inferred from the distribution of mitochondrial haplotypes

The genetic differentiation among populations of the leaf beetle Chrysolina virgata living in wetlands of Japan was studied based on the sequence data of the mitochondrial cytochrome oxidase subunit I gene region (750 bp). Two distinct lineages of mitochondrial haplotypes were found: one (clade A) consisted of 26 haplotypes distributed over the distribution range of C. virgata between north‐east Honshu and Kyushu, whereas the other (clade B) was monotypic and confined to a small region in north‐east Honshu where it coexisted with clade A. Nested clade analysis for these haplotypes suggested that range expansion and following differentiation due to isolation by distance might have resulted in the present distribution pattern of the haplotypes in clade A. We discuss the evolutionary process leading to the occurrence of two distinct haplotype clades in Japan in terms of repeated colonization from the continent and range expansion and contraction during climatic changes.     

Mitochondrial DNA Variation and the Evolution of Robertsonian Chromosomal Races of House Mice, Mus Domesticus

The house mouse, Mus domesticus, includes many distinct Robertsonian (Rb) chromosomal races with diploid numbers from 2n = 22 to 2n = 38. Although these races are highly differentiated karyotypically, they are otherwise indistinguishable from standard karyotype (i.e., 2n = 40) mice, and consequently their evolutionary histories are not well understood. We have examined mitochondrial DNA (mtDNA) sequence variation from the control region and the ND3 gene region among 56 M. domesticus from Western Europe, including 15 Rb populations and 13 standard karyotype populations, and two individuals of the sister species, Mus musculus. mtDNA exhibited an average sequence divergence of 0.84% within M. domesticus and 3.4% between M. domesticus and M. musculus. The transition/transversion bias for the regions sequenced is 5.7:1, and the overall rate of sequence evolution is approximately 10% divergence per million years. The amount of mtDNA variation was as great among different Rb races as among different populations of standard karyotype mice, suggesting that different Rb races do not derive from a single recent maternal lineage. Phylogenetic analysis of the mtDNA sequences resulted in a parsimony tree which contained six major clades. Each of these clades contained both Rb and standard karyotype mice, consistent with the hypothesis that Rb races have arisen independently multiple times. Discordance between phylogeny and geography was attributable to ancestral polymorphism as a consequence of the recent colonization of Western Europe by mice. Two major mtDNA lineages were geographically localized and contained both Rb and standard karyotype mice. The age of these lineages suggests that mice have moved into Europe only within the last 10,000 years and that Rb populations in different geographic regions arose during this time.     

Phylogeny,molecular ecology and taxonomy of southern Iberian lineages of <Emphasis Type="Italic">Triops mauritanicus</Emphasis> (Crustacea: Notostraca)

We investigated the phylogeography of the main lineages in the tadpole shrimp Triops mauritanicus Ghigi in the south-western Iberian Peninsula, using mitochondrial 12S and 16S rDNA sequences. Our results indicate that a fourth, hitherto unknown main phylogenetic lineage occurs in Iberia, so that in total, the species is divided into six distinct clades, comprising T. m. mauritanicus, T. m. simplex Ghigi, and four as yet unnamed lineages that appear to be endemic to Iberia. Percentages of sequence divergence among the main clades in T. mauritanicus reach the range reported for recognized species in other notostracan lineages. A thorough morphological investigation also revealed that the differentiation among these lineages is higher than previously thought, and that populations of three of the main clades within T. mauritanicus can be reliably separated from each other and from the remaining lineages based on the morphology of adult males. The remaining clades also show a significant level of morphological differentiation, but include a certain proportion of populations for which the additional application of molecular methods is needed for a reliable determination. The geographic distributions of 12S haplotypes are indicative of frequent dispersal events and gene flow among populations belonging to the same main lineage, but give no evidence of recent migration events among different main lineages, suggesting that there is no gene flow among the latter. Our data thus suggest that the six main lineages within T. mauritanicus represent distinct species. We therefore describe the Iberian lineages as T. baeticus Korn n. sp., T. emeritensis Korn & Pérez-Bote n. sp., T. gadensis Korn & García-de-Lomas n. sp., and T. vicentinus Korn, Machado, Cristo & Cancela da Fonseca n. sp., and reinstate T. simplex Ghigi to full species status. Our data confirm the general, previously recognized pattern of a lower dispersal probability in gonochoric Triops taxa. However, we found evidence that passive dispersal in Triops may be further complicated by a strong habitat dependence of dispersal probability, mediated by prevailing dispersal vectors.     

Cryptic divergence and strong population structure in the colonial invertebrate Pycnoclavella communis (Ascidiacea) inferred from molecular data

We studied sequence variation in the mitochondrial gene cytochrome c oxidase subunit I (COI) for 135 individuals from eight Mediterranean populations of the colonial ascidian Pycnoclavella communis across most of its presently known range of distribution in the Mediterranean. Three haplotypes from Atlantic locations were also included in the study. Phylogenetic, phylogeographic and population genetic analyses were used to unravel the genetic variability within and between populations. The study revealed 32 haplotypes for COI, 29 of them grouped within two Mediterranean lineages of P. communis (mean nucleotide divergence between lineages was 8.55%). Phylogenetic and network analyses suggest the possible existence of cryptic species corresponding to these two lineages. Population genetic analyses were restricted to the five populations belonging to the main genetic lineage, and for these localities we compared the information gleaned from COI sequence data and from eight microsatellite loci. A high genetic divergence between populations was substantiated using both kinds of markers (COI, global Fst=0.343; microsatellite loci, global Fst=0.362). There were high numbers of private haplotypes (COI) and alleles (microsatellites) in the populations studied. Restricted gene flow and inbreeding occur in the present range of distribution of the species. Microsatellite loci showed a strong incidence of failed amplifications, which we attribute to the marked intraspecies variability that hampered the application of these highly specific markers. Our results show important genetic variability at all levels studied, from within populations to between basins, possibly coupled to speciation processes. This variability is attributable to restricted gene flow among populations due to short-distance dispersal of the larvae.     

Spatial genetic variation, phylogeography and barcoding of the peritrichous ciliate Carchesium polypinum

Most recent studies of geographic distribution of microbial eukaryotes have focused on marine rather than freshwater protists. Here, we used the freshwater peritrich ciliate Carchesium polypinum to quantify the degree of genetic diversity of four closely related and previously described lineages and to determine whether patterns of genetic differentiation showed geographic partitioning. Using an expanded dataset of 100 isolates and employing the mitochondrial marker cytochrome oxidase c subunit I (cox-1), we enriched the 6 previously identified clades of Carchesium polypinum. We found a large degree of geographic overlap among the different clades (e.g. to the level of range of sampling), but also a spatially restricted clade (e.g. to the level of one river basin). Furthermore, we present evidence of a clear geographic separation in one of the lineages with Canadian and North Carolinian isolates grouping in two distinct clusters.     

Structural features and evolutionary considerations of group IB introns in SSU rDNA of the lichen fungus Teloschistes

Different species of the lichen-forming ascomycete fungus Teloschistes were found to contain group IB introns at position S1506 in the small subunit ribosomal RNA gene. We have characterized the structural organization and phylogeny of the Teloschistes introns Tco.S1506, Tla.S1506, and Tvi.S1506. Common features to all the introns are a small size, a compact RNA structure, and an atypical catalytic ribozyme core sequence motif. Variations in intron sizes, due to sequence extensions in the P1 and P8 loop segments, were observed in different species and isolates. Phylogenetic analyses based on the ITS1-5.8S-ITS2 region as well as the introns show that the Teloschistes S1506 introns represent a distinct evolutionary isolated cluster among the nuclear group I introns. Furthermore, introns from different lineages of Teloschistes villosus appear not strictly vertically inherited probably due to horizontal transfer in one of the lineages.     

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre

High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in ∼60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia -related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.