Morphology and gene sequence of Levicoleps biwae n. gen., n. sp. (Ciliophora, Prostomatida), a proposed endemic from the ancient Lake Biwa, Japan

Levicoleps biwae n. gen., n. sp. was discovered in organic mud on the shore of Lake Biwa, Japan. Its morphology and small subunit rRNA gene sequence were studied with standard methods. Further, we established a terminology for the colepid armour and selected four features for genus recognition: the number of armour tiers, the structure of the tier plates, the presence/absence of armour spines, and the number of adoral organelles (three or five). The Japanese colepid, a barrel-shaped ciliate with an average size of 75 x 45 microm, has six armour tiers and hirtus-type tier plates, but lacks armour spines, both in the environment and in laboratory culture. Thus, it is considered to represent a new genus. This rank is supported by the considerable genetic distance (7%) from the common Coleps hirtus. Although L. biwae looks quite similar to C. hirtus in vivo, it is very likely most closely related to Coleps amphacanthus, a species with conspicuous armour spines, as indicated by body size, the number of ciliary rows and, especially, the multiple caudal cilia. Lake Biwa is about four million years old and inhabited by many endemic organisms, ranging from algae to large fish. Thus, we suspect that L. biwae is restricted to Lake Biwa or, at least, to Asia. Based on literature data and the generic features established, we also propose the new genus Reticoleps for Coleps remanei Kahl, 1933, and resurrect the genus Pinacocoleps Diesing, 1865 to include Coleps incurvus Ehrenberg, 1833, Coleps pulcher Spiegel, 1926, Coleps tessalatus Kahl, 1930 and, probably, Baikalocoleps quadratus Obolkina, 1995a. Nine colepid genera are diagnosed and dichotomously keyed.     

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.     

GENE FLOW AND SPECIES DELIMITATION: A CASE STUDY OF TWO PINE SPECIES WITH OVERLAPPING DISTRIBUTIONS IN SOUTHEAST CHINA

Species delimitation detected by molecular markers is complicated by introgression and incomplete lineage sorting between species. Recent modeling suggests that fixed genetic differences between species are highly related to rates of intraspecific gene flow. However, it remains unclear whether such differences are due to high levels of intraspecific gene flow overriding the spread of introgressed alleles or favoring rapid lineage sorting between species. In pines, chloroplast (cp) and mitochondrial (mt) DNAs are normally paternally and maternally inherited, respectively, and thus their relative rates of intraspecific gene flow are expected to be high and low, respectively. In this study, we used two pine species with overlapping geographical distributions in southeast China, P. massoniana and P. hwangshanensis, as a model system to examine the association between organelle gene flow and variation within and between species. We found that cpDNA variation across these two pine species is more species specific than mtDNA variation and almost delimits taxonomic boundaries. The shared mt/cp DNA genetic variation between species shows no bias in regard to parapatric versus allopatric species’ distributions. Our results therefore support the hypothesis that high intraspecific gene flow has accelerated cpDNA lineage sorting between these two pine species.     

Comprehensive DNA barcode coverage of North American birds

DNA barcoding seeks to assemble a standardized reference library for DNA-based identification of eukaryotic species. The utility and limitations of this approach need to be tested on well-characterized taxonomic assemblages. Here we provide a comprehensive DNA barcode analysis for North American birds including 643 species representing 93% of the breeding and pelagic avifauna of the USA and Canada. Most (94%) species possess distinct barcode clusters, with average neighbour-joining bootstrap support of 98%. In the remaining 6%, barcode clusters correspond to small sets of closely related species, most of which hybridize regularly. Fifteen (2%) currently recognized species are comprised of two distinct barcode clusters, many of which may represent cryptic species. Intraspecific variation is weakly related to census population size and species age. This study confirms that DNA barcoding can be effectively applied across the geographical and taxonomic expanse of North American birds. The consistent finding of constrained intraspecific mitochondrial variation in this large assemblage of species supports the emerging view that selective sweeps limit mitochondrial diversity.     

Ecogeographic variation of body size in the spectacled salamanders (Salamandrina): influence of genetic structure and local factors

Aim The patterns and causes of ecogeographical body size variation in ectotherms remain controversial. In amphibians, recent genetic studies are leading to the discovery of many cryptic species. We analysed the relationships between body size and climate for a salamander (Salamandrina) that was recently separated into two sibling species, to evaluate how ignoring interspecific and intraspecific genetic structure may affect the conclusions of ecogeographical studies. We also considered the potential effects of factors acting at a local scale. Location Thirty‐four populations covering the whole range of Salamandrina, which is endemic to peninsular Italy. Methods We pooled original data and data from the literature to obtain information on the snout–vent length (SVL) of 3850 Salamandrina females; we obtained high‐resolution climatic data from the sampled localities. We used an information‐theoretic approach to evaluate the roles of climate, genetic features (mitochondrial haplogroup identity) and characteristics of aquatic oviposition sites. We repeated our analyses three times: in the first analysis we ignored genetic data on intraspecific and interspecific variation; in the second one we considered the recently discovered differences between the two sibling species; in the third one we included information on intraspecific genetic structure within Salamandrina perspicillata (for Salamandrina terdigitata the sample size was too small to perform intraspecific analyses). Results If genetic information was ignored, our analysis suggested the existence of a relationship between SVL and climatic variables, with populations of large body size in areas with high precipitation and high thermal range. If species identity was included in the analysis, the role of climatic features was much weaker. When intraspecific genetic differences were also considered, no climatic feature had an effect. In all analyses, local factors were important and explained a large proportion of the variation; populations spawning in still water had a larger body size. Main conclusions An imperfect knowledge of species boundaries, or overlooking the intraspecific genetic variation can strongly affect the results of analyses of body size variation. Furthermore, local factors can be more important than the large‐scale parameters traditionally considered, particularly in species with a small range.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Phylogeography and sexual macrocyst formation in the social amoeba <Emphasis Type="Italic">Dictyostelium giganteum</Emphasis>

Background  

Microorganisms are ubiquitous, yet we are only beginning to understand their diversity and population structure. Social amoebae (Dictyostelia) are a diverse group of unicellular eukaryotic microbes that display a unique social behaviour upon starvation in which cells congregate and then some die to help others survive and disperse. The genetic relationships among co-occurring cells have a major influence on the evolution of social traits and recent population genetic analysis found extensive genetic variation and possible cryptic speciation in one dictyostelid species (Dictyostelium purpureum). To further characterize the interplay among genetic variation, species boundaries, social behaviour, and reproductive isolation in the Dictyostelia, we conducted phylogenetic analyses and mating experiments with the geographically widespread social amoeba Dictyostelium giganteum.     

Cryptic Species? Patterns of Maternal and Paternal Gene Flow in Eight Neotropical Bats

Levels of sequence divergence at mitochondrial loci are frequently used in phylogeographic analysis and species delimitation though single marker systems cannot assess bi-parental gene flow. In this investigation I compare the phylogeographic patterns revealed through the maternally inherited mitochondrial COI region and the paternally inherited 7(th) intron region of the Dby gene on the Y-chromosome in eight common Neotropical bat species. These species are diverse and include members of two families from the feeding guilds of sanguivores, nectarivores, frugivores, carnivores and insectivores. In each case, the currently recognized taxon is comprised of distinct, substantially divergent intraspecific mitochondrial lineages suggesting cryptic species complexes. In Chrotopterus auritus, and Saccopteryx bilineata I observed congruent patterns of divergence in both genetic regions suggesting a cessation of gene flow between intraspecific groups. This evidence supports the existence of cryptic species complexes which meet the criteria of the genetic species concept. In Glossophaga soricina two intraspecific groups with largely sympatric South American ranges show evidence for incomplete lineage sorting or frequent hybridization while a third group with a Central American distribution appears to diverge congruently at both loci suggesting speciation. Within Desmodus rotundus and Trachops cirrhosus the paternally inherited region was monomorphic and thus does not support or refute the potential for cryptic speciation. In Uroderma bilobatum, Micronycteris megalotis and Platyrrhinus helleri the gene regions show conflicting patterns of divergence and I cannot exclude ongoing gene flow between intraspecific groups. This analysis provides a comprehensive comparison across taxa and employs both maternally and paternally inherited gene regions to validate patterns of gene flow. I present evidence for previously unrecognized species meeting the criteria of the genetic species concept but demonstrate that estimates of mitochondrial diversity alone do not accurately represent gene flow in these species and that contact/hybrid zones must be explored to evaluate reproductive isolation.     

Isolation of microsatellite loci from the scleractinian corals,Montastraea cavernosa and Porites astreoides

The ability to assess genetic variation is critical for determining genetic diversity and population structure. In corals, slow evolutionary rates in mitochondrial genomes have left allozymes as the only markers presently available to investigate patterns of intraspecific genetic variation. Characteristics of microsatellites render them more informative than allozymes for such analyses; however, few coral microsatellites are available. This study describes polymorphic microsatellite loci isolated from two scleractinian coral species. Most loci exhibit significant heterozygote deficiencies, likely due to nonrandom mating or Wahlund effects. These markers are being used to investigate gene flow among populations, providing insight into reef connectivity.     

A role for gene duplication and natural variation of gene expression in the evolution of metabolism

Background

Most eukaryotic genomes have undergone whole genome duplications during their evolutionary history. Recent studies have shown that the function of these duplicated genes can diverge from the ancestral gene via neo- or sub-functionalization within single genotypes. An additional possibility is that gene duplicates may also undergo partitioning of function among different genotypes of a species leading to genetic differentiation. Finally, the ability of gene duplicates to diverge may be limited by their biological function.

Methodology/Principal Findings

To test these hypotheses, I estimated the impact of gene duplication and metabolic function upon intraspecific gene expression variation of segmental and tandem duplicated genes within Arabidopsis thaliana. In all instances, the younger tandem duplicated genes showed higher intraspecific gene expression variation than the average Arabidopsis gene. Surprisingly, the older segmental duplicates also showed evidence of elevated intraspecific gene expression variation albeit typically lower than for the tandem duplicates. The specific biological function of the gene as defined by metabolic pathway also modulated the level of intraspecific gene expression variation. The major energy metabolism and biosynthetic pathways showed decreased variation, suggesting that they are constrained in their ability to accumulate gene expression variation. In contrast, a major herbivory defense pathway showed significantly elevated intraspecific variation suggesting that it may be under pressure to maintain and/or generate diversity in response to fluctuating insect herbivory pressures.

Conclusion

These data show that intraspecific variation in gene expression is facilitated by an interaction of gene duplication and biological activity. Further, this plays a role in controlling diversity of plant metabolism.     

Spatial Genetic Structure and Mitochondrial DNA Phylogeography of Argentinean Populations of the Grasshopper Dichroplus elongatus

Many grasshopper species are considered of agronomical importance because they cause damage to pastures and crops. Comprehension of pest population dynamics requires a clear understanding of the genetic diversity and spatial structure of populations. In this study we report on patterns of genetic variation in the South American grasshopper Dichroplus elongatus which is an agricultural pest of crops and forage grasses of great economic significance in Argentina. We use Direct Amplification of Minisatellite Regions (DAMD) and partial sequences of the cytochrome oxydase 1 (COI) mitochondrial gene to investigate intraspecific structure, demographic history and gene flow patterns in twenty Argentinean populations of this species belonging to different geographic and biogeographic regions. DAMD data suggest that, although genetic drift and migration occur within and between populations, measurable relatedness among neighbouring populations declines with distance and dispersal over distances greater than 200 km is not typical, whereas effective gene flow may occur for populations separated by less than 100 km. Landscape analysis was useful to detect genetic discontinuities associated with environmental heterogeneity reflecting the changing agroecosystem. The COI results indicate the existence of strong genetic differentiation between two groups of populations located at both margins of the Paraná River which became separated during climate oscillations of the Middle Pleistocene, suggesting a significant restriction in effective dispersion mediated by females and large scale geographic differentiation. The number of migrants between populations estimated through mitochondrial and DAMD markers suggest that gene flow is low prompting a non-homogeneous spatial structure and justifying the variation through space. Moreover, the genetic analysis of both markers allows us to conclude that males appear to disperse more than females, reducing the chance of the genetic loss associated with recent anthropogenic fragmentation of the D. elongatus studied range.     

In situ nutrient enrichment experiments with periphyton in agricultural streams

The abundance, composition and seasonal distribution of planktonic protozoans and physical and chemical variables such as temperature, dissolved oxygen content, pH, and chlorophyll a concentration were analyzed monthly from February 1985 to January 1986 in eutrophic Rio Grande Reservoir. Analyses were performed on water samples collected at the surface and bottom (0.5 m above the sediment) in three stations, and individual distribution profiles were constructed for the major species. Dominant protozoan taxa included Codonella cratera, Coleps amphacantus, Coleps hirtus, Halteria grandinella, Mesodinium pulex, Tintinnidium sp., Urotricha spp., Vorticella spp., and Difflugia sp.. The distribution of some species was probably related to temperature and dissolved oxygen content.     

Assessing the use of mitochondrial cox1 gene and cox2-3 spacer for genetic diversity study of Malaysian Gracilaria changii (Gracilariaceae, Rhodophyta) from Peninsular Malaysia

Advances in DNA-based genetic markers provide the essential tools in measurement of genetic diversity relating to the evolution, biogeography, and systematics of red algae by exploiting genetic variation in the entire genome of organisms. The understanding of genetic diversity in Gracilaria changii (Gracilariaceae, Rhodophyta) will provide valuable information for conservation, plant breeding management, and strain selection for cultivation. However, information of intraspecific genetic variation is still rudimentary. In this study, two mitochondrial encoded markers, cytochrome oxidase subunit 1 (cox1) and intergenic spacer between the cytochrome oxidase subunits 2 and 3 (cox2-3 spacer) were used to investigate genetic diversity in 40 individuals of G. changii collected from 11 different geographically distinct populations from Peninsular Malaysia. Seven distinct mitochondrial haplotypes were identified with the cox1 gene and three mitochondrial haplotypes with the cox2-3 spacer. Intraspecific nucleotide differences ranged from 0 to 6 bp for the cox1 and 0–4 bp for the cox2-3 spacer, respectively. This is the first report comparing the suitability of mitochondrial markers of the cox1 gene and the cox2-3 spacer for genetic diversity studies on G. changii. The present study showed that the cox1 gene is a potential molecular marker to infer intraspecific genetic variation in red macroalgae. The cox1 marker is more variable compared to the cox2-3 spacer and revealed genetic variation and phylogeographic structure for this ecologically and economically important species.     

DNA barcoding and genetic diversity of phyllostomid bats from the Yucatan Peninsula with comparisons to Central America

The mitochondrial cytochrome c oxidase subunit I gene is the standard DNA barcoding region used for species identification and discovery. We examined the variation of COI (454 bp) to discriminate 20 species of bats in the family Phyllostomidae that are found in the Yucatan Peninsula of southeastern Mexico and northern Guatemala and compared them genetically to other samples from Central America. The majority of these species had low intraspecific variation (mean = 0.75%), but some taxa had intraspecific variation ranging to 8.8%, suggesting the possibility of cryptic species (i.e. Desmodus rotundus and Artibeus jamaicensis). There was a recurring biogeographic pattern in eight species with a separation of northern and southern Middle American localities. The Yucatan Peninsula was a discrete area identified in four species, whereas Panama was recovered in five species of phyllostomid bats. Our study establishes a foundation for further molecular work incorporating broader taxonomic and geographic coverage to better understand the phylogeography and genetic diversity that have resulted from the ecological constraints in this region and the remarkable differentiation of bats in the Neotropics.     

Phylogeography and support vector machine classification of colour variation in panther chameleons

Lizards and snakes exhibit colour variation of adaptive value for thermoregulation, camouflage, predator avoidance, sexual selection and speciation. Furcifer pardalis, the panther chameleon, is one of the most spectacular reptilian endemic species in Madagascar, with pronounced sexual dimorphism and exceptionally large intraspecific variation in male coloration. We perform here an integrative analysis of molecular phylogeography and colour variation after collecting high‐resolution colour photographs and blood samples from 324 F. pardalis individuals in locations spanning the whole species distribution. First, mitochondrial and nuclear DNA sequence analyses uncover strong genetic structure among geographically restricted haplogroups, revealing limited gene flow among populations. Bayesian coalescent modelling suggests that most of the mitochondrial haplogroups could be considered as separate species. Second, using a supervised multiclass support vector machine approach on five anatomical components, we identify patterns in 3D colour space that efficiently predict assignment of male individuals to mitochondrial haplogroups. We converted the results of this analysis into a simple visual classification key that can assist trade managers to avoid local population overharvesting.     

Monomorphic species specific DNA, detected by polymerase chain reaction with random primers

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.     

Species-Specific Random Amplified Monomorphic DNA

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.     

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae)

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.     

Patterns of genetic variation in Pacific island land snails: the distribution of cytochrome b lineages among Society Island Partula

The radiation of Partula land snails has produced a large array of distinct morphological, ecological and behavioural types occupying many tropical volcanic islands in the Pacific Ocean. Within the Society Islands of French Polynesia, the mode of evolution is thought to have involved a single colonization event on each island, with later speciation occurring largely in situ. The present study examines genetic variation in the mitochondrial cytochrome b gene among taxa within the Society Island archipelago. Levels of intraspecific variation are found to be high, but variation among species is sometimes small. Mitochondrial variants do not always cluster according to island and some species are found to be polyphyletic in the cytochrome b tree, despite other morphological and molecular evidence that strongly supports their monophyly. A possible explanation for the polyphyly of species is that different variants are derived from ancestral mitochondrial polymorphisms that have been retained despite speciation events. Although it is possible that there has been some gene flow among islands, the distribution of mitochondrial lineages across islands strongly indicates that their origins predate the colonization of the islands in the study, and that they are very unlikely to have evolved entirely in situ.     

Complete mitochondrial genomes of Diplogonoporus balaenopterae and Diplogonoporus grandis (Cestoda: Diphyllobothriidae) and clarification of their taxonomic relationships

Although the diplogonadic human tapeworm, Diplogonoporus grandis, has long been considered to be a synonym of the whale tapeworm, Diplogonoporus balaenopterae, the identity of the both species at the complete mitochondrial genomes and nuclear DNA levels has been not sufficiently undertaken to date. In the present study, to clarify the taxonomic relationships between D. balaenopterae and D. grandis at the molecular level, the complete mitochondrial genomes of both species were sequenced and compared. In addition, the genetic variation in the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the nuclear internal transcribed spacer-1 (ITS-1) region of the ribosomal RNA gene were examined. The complete mitochondrial genomes of D. balaenopterae and D. grandis consisted of 13,724 bp and 13,725 bp, respectively. These mitochondrial genomes contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes and two longer non-coding regions. Except for Hymenolepis diminuta, the genomic organization in both species was essentially identical to that in other cestode genomes examined to date. However, differences were observed between Diplogonoporus and Diphyllobothrium species in abbreviated stop codons, sequences and the number of repeat units in the 2nd non-coding regions. The genetic differences observed in the mitochondrial genomes, cox1 and ITS-1 regions of both species were considered typical of intraspecific variation. In conclusion, D. balaenopterae is a taxonomically valid species and D. grandis is a junior synonym of D. balaenopterae based on the zoological nomenclature. Further, molecular-phylogenetic analysis confirmed that D. balaenopterae is more closely related to Diphyllobothrium stemmacephalum, the type-species of the genus Diphyllobothrium, and the taxonomical validity of the genera Diplogonoporus and Diphyllobothrium was also discussed.