Mitochondrial DNA sequence variation in the spruce budworm species complex (Choristoneura: Lepidoptera)

A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.      

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Phylogeography of a specialist insect, Adelges cooleyi: historical and contemporary processes shape the distribution of population genetic variation

Adelges cooleyi is a host-alternating, gall-making insect native to the Rocky Mountains and Cascade Mountains in western North America. The insect's primary hosts are Picea (spruce) species, and its secondary host is Pseudotsuga menziesii , Douglas fir. To determine whether there are large-scale patterns of genetic variation in this specialist insect, we created molecular phylogenies of geographically separate samples of A. cooleyi using sequence data from two mitochondrial (mtDNA) genes and amplified fragment length polymorphisms (AFLPs). Three divergent mtDNA lineages were identified. Analysis of mtDNA and AFLP genetic variation revealed that samples from southeastern Arizona are genetically isolated from all other samples. AFLP data identified possible gene flow between individuals from divergent mtDNA lineages in an area in the central Rocky Mountains. Factors that likely affected divergence within A. cooleyi were identified by comparing our conclusions with well-known changes in the distribution of vegetation in response to glaciations and previous phylogeographical work conducted on this specialist insect's host-plants. In addition to documenting previously unknown patterns of genetic variation in A. cooleyi , our work provides the basis for a testable hypothesis regarding the extent to which the distribution of variation in Picea and Pseudotsuga hosts mediates the distribution of genetic variation for this specialist insect.     

Two mitochondrial lineages in Korean freshwater Corbicula (Corbiculidae: bivalvia)

The Korean freshwater Corbicula was surveyed genetically by sequencing 614-bp homologous fragment of mitochondrial cytochrome oxidase I subunit. Among a total of 127 individuals collected from 12 Korean freshwater localities we found only two COI haplotypes and these differed by a total of 9 base substitutions. Although the sequence divergence between the two haplotypes is moderate (p = 1.47%), placing the two mitotypic sequences in the context of Asian mtDNA phylogeny reveals that Korean freshwater Corbicula is comprised of two independent freshwater mitochondrial lineages. These results are in serious disagreement with the long-standing conclusions of earlier conchology-based taxonomic work on Korean Corbicula in which a number of species names (a minimum of 10 nominal species) have been used. This indicates that morphological characteristics alone are poor criteria for species-level identification in this group. In addition, our COI dataset shows that there is an extremely low level of genetic variation in Korean freshwater populations, suggesting that these populations have passed through a severe population bottleneck that greatly reduced their genetic variability. Our data also provide new information on the biogeographic distribution of Korean freshwater Corbicula. When haplotypic frequencies were compared, it was evident that the two Korean freshwater mitochondrial lineages have achieved very different distribution ranges: the predominant lineage (FWKR1) is widely distributed in Korean freshwater systems, whereas the minor lineage (FWKR2) is restricted to a relatively narrow range.     

Delimiting cohesion species: extreme population structuring and the role of ecological interchangeability

Species exhibiting morphological homogeneity and strong population structuring present challenging taxonomic problems: morphology-based approaches infer few species, whereas genetic approaches often indicate more. Morphologically cryptic, yet genetically divergent species groups require alternative approaches to delimiting species that assess adaptive divergence and ecological interchangeability of lineages. We apply such an approach to Promyrmekiaphila, a small genus (three nominal taxa) of trapdoor spiders endemic to northern California to define cohesion species (lineages that are genetically exchangeable and ecologically interchangeable). Genetic exchangeability is evaluated using standard phylogeographical techniques (e.g. nested clade analysis); ecological interchangeability is assessed using two GIS-based approaches. First, climatic values are extracted from layer data for each locality point and utilized in a principal components analysis followed by MANOVA. Second, niche-based distribution models of genetically divergent lineages are created using a maximum-entropy modelling approach; the amount of overlap among lineages is calculated and evaluated against a probability distribution of null overlap. Lineages that have significant amounts of predicted overlap are considered ecologically interchangeable. Based on a synthetic evaluation of ecological interchangeability, geographical concordance, and morphological differentiation, we conclude that Promyrmekiaphila comprises six cohesion species, five of which are cryptic (i.e. undetectable by conventional means).     

Simple identification of mitochondrial lineages in contact zones based on lineage-selective primers

A variety of research projects focus on genetic variation among and within maternal lineages as encompassed by mitochondrial DNA (mtDNA). While mtDNA often differs substantially between species, large differences may also be found within species. The evaluation of such divergent lineages, for example in intraspecific contact zones (hybrid zones), commonly involves sequencing numerous individuals. Large‐scale sequencing is both expensive and labour‐intensive. Based on sequences from 15 individuals, we devised a simple and quick polymerase chain reaction assay for identification of divergent mtDNA lineages in a secondary contact zone of the side‐blotched lizard (Uta stansburiana). The application uses lineage‐selective primers to amplify a lineage‐diagnostic product, and is based on each group of mtDNA haplotypes being a monophyletic assemblage of haplotypes sharing the same maternal ancestry, deeply divergent from the other group. The assay was tested on a larger sample (n = 147) of specimens from the contact zone, confirming its usefulness in quick and reliable identification of mtDNA lineages. This approach can be modified for other species, provided diagnostic lineage variation is available, and may also be performed in simple laboratory settings while conducting fieldwork.     

Genetic surfing,not allopatric divergence,explains spatial sorting of mitochondrial haplotypes in venomous coralsnakes

Strong spatial sorting of genetic variation in contiguous populations is often explained by local adaptation or secondary contact following allopatric divergence. A third explanation, spatial sorting by stochastic effects of range expansion, has been considered less often though theoretical models suggest it should be widespread, if ephemeral. In a study designed to delimit species within a clade of venomous coralsnakes, we identified an unusual pattern within the Texas coral snake (Micrurus tener): strong spatial sorting of divergent mitochondrial (mtDNA) lineages over a portion of its range, but weak sorting of these lineages elsewhere. We tested three alternative hypotheses to explain this pattern—local adaptation, secondary contact following allopatric divergence, and range expansion. Collectively, near panmixia of nuclear DNA, the signal of range expansion associated sampling drift, expansion origins in the Gulf Coast of Mexico, and species distribution modeling suggest that the spatial sorting of divergent mtDNA lineages within M. tener has resulted from genetic surfing of standing mtDNA variation—not local adaptation or allopatric divergence. Our findings highlight the potential for the stochastic effects of recent range expansion to mislead estimations of population divergence made from mtDNA, which may be exacerbated in systems with low vagility, ancestral mtDNA polymorphism, and male‐biased dispersal.     

Molecular systematics and phylogeography of Amazonian poison frogs of the genus Dendrobates

The study of Amazonian biodiversity requires detailed knowledge of the phylogenetic relationships of closely related taxa distributed across Amazonia. The Amazonian poison frogs of the genus Dendrobates have undergone many taxonomic revisions, but the phylogenetic relationships within this group remain poorly understood. Most previous classifications were based on morphology and skin toxin analyses, with limited use of DNA sequence data. Using mtDNA sequence data from four gene regions (cytochrome b, cytochrome oxidase I, 16S rRNA, and 12S rRNA), we present a molecular phylogenetic analysis of the evolutionary relationships within a representative group of Amazonian Dendrobates. We use the resulting phylogenetic hypothesis to investigate different biogeographic hypotheses concerning genetic divergence and species diversity in Amazonia. The results of the analysis support the presence of ancient paleogeographic barriers to gene flow between eastern and western Amazonia, and indicate substantial genetic divergence between species found in the northern and southern regions of western Amazonia.     

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.     

Biogeographic patterns and current distribution of molecular-genetic variation among populations of speckled dace, Rhinichthys osculus (Girard)

We examined genetic diversity within- and among-populations of speckled dace (Rhinichthys osculus) in five major drainage systems in the state of Oregon in western North America. Analysis of sequence variation in a 670-bp segment of the mitochondrial cytochrome b gene revealed deep divergence among basins and high genetic diversity within basins. Application of a molecular clock indicated that the divergence time among basins reflects vicariant events during the late Miocene to early Pliocene. The high levels of genetic diversity observed within basins is likely due to large historic population sizes, in particular, within the Klamath Basin. Two highly divergent mtDNA lineages were found to co-occur in populations in the Klamath Basin. This result may be indicative of a complex history of isolation and reconnection in this basin and/or multiple colonization events. Based on the observed level of mtDNA divergence these lineages may represent two reproductively isolated sympatric taxa. We recommend that major basins be regarded as distinct ESUs based on high levels of subdivision, deep divergences, and reciprocal monophyly among basins.     

Cryptic speciation in the Anadenobolus excisus millipede species complex on the Island of Jamaica

Anadenobolus excisus is a large species of millipede endemic to the Caribbean Island of Jamaica. Initial detailed morphological studies showed little or no discrete variation across this species' distribution in somatic or, in particular. genitalic morphology. However, a molecular survey based on approximately 1000 base pairs of the mitochondrial (mtDNA) 16S rRNA gene that examines 242 individuals sampled from 54 localities reveals three highly divergent mtDNA lineages. A lack of discrete morphological differentiation suggests that genetic and morphological divergence may be decoupled, a pattern inconsistent with a number of evolutionary models. In contrast to minimal morphological divergence, size variation among mtDNA lineages suggests that character displacement has occurred and that these lineages are cohesive in sympatry. We conclude that A. excisus is actually a complex of three cryptic species and that morphological approaches to delineating millipede species may sometimes underestimate evolutionary diversity.     

Cryptic diversity in European bats

Different species of bat can be morphologically very similar. In order to estimate the amount of cryptic diversity among European bats we screened the intra- and interspecific genetic variation in 26 European vespertilionid bat species. We sequenced the DNA of subunit 1 of the mitochondrial protein NADH dehydrogenase (ND1) from several individuals of a species, which were sampled in a variety of geographical regions. A phylogeny based on the mitochondrial (mt) DNA data is in good agreement with the current classification in the family. Highly divergent mitochondrial lineages were found in two taxa, which differed in at least 11% of their ND1 sequence. The two mtDNA lineages in Plecotus austriacus correlated with the two subspecies Plecotus austriacus austriacus and Plecotus austriacus kolombatovici. The two mtDNA lineages in Myotis mystacinus were partitioned among two morphotypes. The evidence for two new bat species within Europe is discussed. Convergent adaptive evolution might have contributed to the morphological similarity among distantly related species if they occupy similar ecological niches. Closely related species may differ in their ecology but not necessarily in their morphology. On the other hand, two morphologically clearly different species (Eptesicus serotinus and Eptesicus nilssonii) were found to be genetically very similar. Neither morphological nor mitochondrial DNA sequence analysis alone can be guaranteed to identify species.     

Phylogeography of spruce beetles (Dendroctonus rufipennis Kirby) (Curculionidae: Scolytinae) in North America

Tree-feeding insects that are widespread in north temperate regions are excellent models for studying how past glaciations have impacted differentiation and speciation. We used mitochondrial DNA (mtDNA) sequences and allele frequencies at nine microsatellite loci to examine genetic population structure across the current range of the spruce beetle (Dendroctonus rufipennis), an economically important insect in North America. Two major haplotype groups occur across northern North America, from Newfoundland to Alaska, on white spruce (Picea glauca), and a third distinctive haplotype group occurs throughout the Rocky Mountains on Engelmann spruce (Picea engelmannii). The two mtDNA lineages found in northern populations are 3-4% divergent from each other and from the lineages found in the Rocky Mountains. Analyses of microsatellite data also suggest the existence of major population groupings associated with different geographical regions. In the Pacific Northwest, concordant contact zones for genetically distinct populations of spruce beetles and their principal hosts appear to reflect recent secondary contact. Although we could detect no evidence of historical mtDNA gene flow between allopatric population groups, patterns of variation in the Pacific Northwest suggest recent hybridization and introgression. Together with the pollen record for spruce, they also suggest that beetles have spread from at least three glacial refugia. A minimum estimate of divergence time between the Rocky Mountain and northern populations was 1.7 Myr (million years), presumably reflecting the combined effects of isolation during multiple glacial cycles.     

Cryptic diversity in a Eurasian water snake (Natrix tessellata, Serpentes: Colubridae): Evidence from mitochondrial sequence data and nuclear ISSR-PCR fingerprinting

The dice snake, Natrix tessellata (Laurenti, 1768), is a suitable study organism to address questions of Eurasian phylogeography due to its wide Palearctic distribution. We analysed complete mitochondrial cytochrome b sequences and nuclear ISSR-PCR fingerprints of more than 300 specimens representing nearly the entire geographic range. Nine major mitochondrial lineages were discovered based on mtDNA sequences. The three most basal lineages comprised populations from Iran, Jordan–Egypt, and Greece, respectively. Other lineages were associated with samples from the Turkish peninsula, the Caucasus, the Aral Sea, and eastern Kazakhstan. A sister-group relationship was found between two lineages from Crete and the European mainland. Assuming an evolutionary rate of 1.35% sequence divergence per million years, among-lineage p-distances of 1.7–8.4% suggest that intraspecific differentiation might date back as far as the Miocene/Pliocene transition 5–6 million years ago. The pattern of genetic differentiation in mitochondrial phylogeny with regard to Asia Minor and the region of the Aral Sea was not congruent with the results of the nuclear ISSR-PCR analyses, and suggests admixing within some mtDNA clades at contact zones. The taxonomic implications of the high intraspecific variation in the dice snake are discussed.     

Complex patterns of genetic and phenotypic divergence in an island bird and the consequences for delimiting conservation units

Substantial phenotypic and genetic variation is often found below the species level and this may be useful in quantifying biodiversity and predicting future diversification. However, relatively few studies have tested whether different aspects of intraspecific variation show congruent patterns across populations. Here, we quantify several aspects of divergence between 13 insular populations of an island endemic bird, the Vanuatu white-eye ( Zosterops flavifrons ). The components of divergence studied are mitochondrial DNA (mtDNA), nuclear DNA microsatellites and morphology. These different aspects of divergence present subtly different scenarios. For instance, an mtDNA phylogenetic tree reveals a potential cryptic species on the most southerly island in Vanuatu and considerable divergence between at least two other major phylogroups. Microsatellite loci suggest that population genetic divergence between insular populations, both between and within phylogroups, is substantial, a result that is consistent with a low level of interisland gene flow. Finally, most populations were found to be strongly morphologically divergent, but no single population was morphologically diagnosable from all others. Taken together, our results show that, although many measures of divergence are concordant in this system, the number of divergent units identified varies widely depending on the characters considered and approach used. A continuum of divergence and a degree of discordance between different characters are both to be expected under simple models of evolution, but they present problems in terms of delimiting conservation units.     

DNA barcoding will often fail to discover new animal species over broad parameter space

With increasing force, genetic divergence of mitochondrial DNA (mtDNA) is being argued as the primary tool for discovery of animal species. Two thresholds of single-gene divergence have been proposed: reciprocal monophyly, and 10 times greater genetic divergence between than within species (the "10x rule"). To explore quantitatively the utility of each approach, we couple neutral coalescent theory and the classical Bateson-Dobzhansky-Muller (BDM) model of speciation. The joint stochastic dynamics of these two processes demonstrate that both thresholds fail to "discover" many reproductively isolated lineages under a single incompatibility BDM model, especially when BDM loci have been subject to divergent selection. Only when populations have been isolated for > 4 million generations did these thresholds achieve error rates of < 10% under our model that incorporates variable population sizes. The high error rate evident in simulations is corroborated with six empirical data sets. These properties suggest that single-gene, high-throughput approaches to discovering new animal species will bias large-scale biodiversity surveys, particularly toward missing reproductively isolated lineages that have emerged by divergent selection or other mechanisms that accelerate reproductive isolation. Because single-gene thresholds for species discovery can result in substantial error at recent divergence times, they will misrepresent the correspondence between recently isolated populations and reproductively isolated lineages (= species).     

Testing paleolimnological predictions with molecular data: the origins of Holarctic Eubosmina

Zooplankton of the family Bosminidae have a unique paleolimnological record in many Holarctic lakes that provides a near continuous record of morphological change for thousands of years. If this morphological change could be interpreted reliably, then a rarely achieved direct observation of macroevolution would be feasible. We tested paleolimnological predictions derived from morphological variation found in the genus Eubosmina using mtDNA and nuclear DNA sequence variation from geographically distant Holarctic sites. The mtDNA and nDNA trees were congruent but genetic divergence was inversely associated with morphological divergence. The three most genetically divergent groups belonged to Eubosmina longispina, whose phylogeography and genetic divergence was consistent with glacial vicariance. The genetic evidence also supported the hypothesis that at least two Nearctic species were recent European introductions. Finally, the genetic evidence was consistent with paleolimnology in the finding of several proposed species undergoing rapid morphological evolution and being post-glacially derived from European E. longispina. The results suggested that lacustrine bosminids are susceptible to geographic speciation processes, and that morphological interpretation of diversity in paleolimnology can be markedly improved by genetic studies.     

AFLP analysis shows high incongruence between genetic differentiation and morphology‐based taxonomy in a widely distributed tortoise

Morphology has traditionally been used to diagnose the taxa of various taxonomic ranks. However, there is growing evidence that morphology is not always able to reveal cryptic taxa, and that pronounced morphological variation could reflect phenotypic plasticity rather than evolutionary divergence. Spur‐thighed tortoises (the Testudo graeca complex), distributed in the western Palaearctic region, are characterized by high morphological variability and complicated taxonomy, which are under debate. Previous molecular studies using mainly mitochondrial DNA (mtDNA) sequences have revealed incongruence between genetic differentiation and morphology‐based taxonomy, suggesting that morphological variability is the result of phenotypic plasticity and stabilizing selection, which masks the true genealogies. In the present study, we used a range‐wide sampling and nuclear Amplified fragment length polymorphism (AFLP) markers to investigate genetic differentiation within the T. graeca complex. We found that spur‐thighed tortoises are differentiated into four geographically well‐defined AFLP groups: Balkans–Middle Eastern, western Mediterranean, Caucasian and central‐eastern Iranian. Compared with the distribution of mtDNA lineages, the groups are largely concordant, although the AFLP markers are less sensitive and distinguish fewer groups than do mtDNA sequences. The AFLP groups show an allopatric or parapatric distribution. The AFLP differentiation conflicts with the previously proposed morphology‐based taxonomy of the complex, suggesting that local adaptation to different environmental conditions may have led to the great extent of morphological variation within the same lineages. We propose a re‐evaluation of the taxa that were confirmed genetically using a thorough morphological analysis corrected for phenotypic plasticity. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.