Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers

Background

The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units.

Principal Findings

The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f. fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled from Ethiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations.

Conclusion/Significance

We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme.     

On estimating diversity in species-rich fish assemblages: an example with coral-reef fishes of eastern Indonesia

A new procedure for estimating fish species richness in open or semi-open habitats is presented with unpublished data on the coral-reef fish assemblages of Raja Ampat, eastern Indonesia, known as the centre of marine biodiversity in the western Pacific.     

Role of predators in the early post-settlement demography of coral-reef fishes

Populations with dispersive larvae are often demographically open such that local reproduction and subsequent larval settlement are not linked. Thus, understanding whether and how settlement patterns are established and subsequently modified is central to understanding local demography. Settlement is typically not measured directly, but rather it is estimated by recruitment, which is the observation of new individuals sometime after settlement. At Lizard Island on the Great Barrier Reef, I examined how patterns of recruitment of coral-reef fishes were modified across a range of natural recruit densities in the presence and absence of resident predators. Resident predators decreased recruitment and increased mortality for all species, but these effects varied considerably among species. The effects of predators on recruitment were at least partly due to mortality within 2 days after settlement. At their most extreme, predators caused recruitment failure of several species of butterflyfish. For one species of damselfish (Pomacentrus amboinensis), predators both induced weakly density-dependent mortality and obscured any relationship between recruitment and subsequent abundance, while for another damselfish (Neopomacentrus cyanomos), mortality was density-independent and subsequent abundance was a function of recruitment. These contrasting results may reflect differences in prey behavior. P. amboinensis tended to feed near or within the branches of coral inhabited by resident predators, while N. cyanomos tended to feed higher in the water column above the reefs, and thus farther away from resident predators. These results highlight the speed and extent to which patterns of settlement are modified, indicating that caution should be exercised when attributing patterns of recruitment to patterns of settlement. Tremendous between-species variation in how patterns of recruitment, and presumably settlement, were modified by predation indicates that generalizations or between-species extrapolations about the magnitude of these effects may be unwarranted.     

Cryptic diversity in Indo-Australian rainbowfishes revealed by DNA barcoding: implications for conservation in a biodiversity hotspot candidate

The rainbowfishes of the family Melanotaeniidae represent one of the largest radiations of freshwater fishes from the Indo-Australian archipelago. A total of 75 nominal species have been described, among which several have become very popular among tropical fish hobbyists because of their tendency to form large schools of colourful individuals. Facing habitat loss and competition or predation by introduced species, this group has become a priority in the conservation of ornamental fishes in Indonesia. In this context, several expeditions have been conducted between 2007 and 2010 in Indonesian Papua with the aim to initiate a large-scale survey of the genetic resources in this group. We assessed the diversity of the Papua rainbowfishes with DNA barcoding. We sequenced the mitochondrial COI gene for 350 specimens belonging to 53 nominal species throughout the Indo-Australian archipelago. Unexpected levels of cryptic diversity and endemism were detected since additional cryptic lineages were detected in several watersheds from the Vogelkop and the Lengguru massif. DNA barcoding supports the presence of nearly 30 evolutionary lineages among the 15 nominal species sampled in the Vogelkop and all these lineages are endemic to a single lake or watershed. This result highlights that the diversity of the family has been largely underestimated and urges for the identification of conservation priorities in Papua.     

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3–22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed ≥2.3% sequence divergence between taxa and ≤1.3% within taxa. We developed a PCR-RFLP based method for unequivocally distinguishing amongst the four cryptic European taxa of this subgenus, B. cryptarum, B. lucorum, B. magnus and B. terrestris, and used it to analyse 391 females of the former three species collected across Ireland, all of which could be unambiguously assigned to species. Bombus lucorum was the most widely distributed and abundant of the cryptarum–lucorum–magnus species complex, comprising 56% of individuals, though it was significantly less abundant at higher altitudes (>200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.     

A new twist in TCR diversity revealed by a forbidden alphabeta TCR

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A^u-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the Vα/Vβ interface exert indirect effects on recognition by influencing the Vα/Vβ interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the Vα/Vβ interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.     

Predator diversity and abundance provide little support for the enemies hypothesis in forests of high tree diversity

Predatory arthropods can exert strong top-down control on ecosystem functions. However, despite extensive theory and experimental manipulations of predator diversity, our knowledge about relationships between plant and predator diversity--and thus information on the relevance of experimental findings--for species-rich, natural ecosystems is limited. We studied activity abundance and species richness of epigeic spiders in a highly diverse forest ecosystem in subtropical China across 27 forest stands which formed a gradient in tree diversity of 25-69 species per plot. The enemies hypothesis predicts higher predator abundance and diversity, and concomitantly more effective top-down control of food webs, with increasing plant diversity. However, in our study, activity abundance and observed species richness of spiders decreased with increasing tree species richness. There was only a weak, non-significant relationship with tree richness when spider richness was rarefied, i.e. corrected for different total abundances of spiders. Only foraging guild richness (i.e. the diversity of hunting modes) of spiders was positively related to tree species richness. Plant species richness in the herb layer had no significant effects on spiders. Our results thus provide little support for the enemies hypothesis--derived from studies in less diverse ecosystems--of a positive relationship between predator and plant diversity. Our findings for an important group of generalist predators question whether stronger top-down control of food webs can be expected in the more plant diverse stands of our forest ecosystem. Biotic interactions could play important roles in mediating the observed relationships between spider and plant diversity, but further testing is required for a more detailed mechanistic understanding. Our findings have implications for evaluating the way in which theoretical predictions and experimental findings of functional predator effects apply to species-rich forest ecosystems, in which trophic interactions are often considered to be of crucial importance for the maintenance of high plant diversity.     

Cryptic speciation in a Holarctic passerine revealed by genetic and bioacoustic analyses

There has been much controversy regarding the timing of speciation events in birds, and regarding the relative roles of natural and sexual selection in promoting speciation. Here, we investigate these issues using winter wrens ( Troglodytes troglodytes ), an unusual example of a passerine with a Holarctic distribution. Geographical variation has led to speculation that the western North American form Troglodytes troglodytes pacificus might be a distinct biological species compared to those in eastern North America (e.g. Troglodytes troglodytes hiemalis ) and Eurasia. We located the first known area in which both forms can be found, often inhabiting neighbouring territories. Each male wren in this area sings either western or eastern song, and the differences in song are as distinct in the contact zone as they are in allopatry. The two singing types differ distinctly in mitochondrial DNA sequences and amplified fragment length polymorpism profiles. These results indicate that the two forms are reproductively isolated to a high degree where they co-occur and are therefore separate species. DNA variation suggests that the initial split between the two species occurred before the Pleistocene, quite long ago for sister species in the boreal forest. Surprisingly, the two forms are similar in morphometric traits and habitat characteristics of territories. These findings suggest that sexual selection played a larger role than habitat divergence in generating reproductive isolation, and raise the possibility that there are other such morphologically cryptic species pairs in North America.     

Cryptic diversity and conservation units in the Bahama parrot

Wide application of genetic approaches has enhanced the detection of cryptic diversity, even in historically well-studied organisms. In addition to improving our knowledge of biodiversity, detection of cryptic diversity can have important management implications within imperiled groups, such as the Cuban parrot complex (Amazona leucocephala). Bahama parrots (A. l. bahamensis) were once widespread throughout the archipelago, but are now restricted to the two largest islands (Abaco and Inagua). Mitochondrial DNA-based population genetic and phylogenetic analyses revealed the distinctiveness of the Abaco, Inagua and now extirpated Acklins populations, detecting diagnostic character support and reciprocal monophyly indicative of three phylogenetic species. Congruent results were obtained for the Abaco and Inagua populations based on Bayesian clustering analyses of microsatellite genotypic data. Genetic signatures of demographic contraction were identified on Abaco, but not Inagua. These findings were consistent with lower genetics-based estimates of effective population size on Abaco, as well as the disproportionate human impacts reported on the island relative to Inagua. Overall, our results suggest that the taxonomy of the Cuban parrot complex requires revision and that the conservation status of the Abaco phylogenetic species should be immediately elevated to reflect its historical isolation, recent population decline and continued threats to its persistence.     

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

Cryptic anuran biodiversity in Bangladesh revealed by mitochondrial 16S rRNA gene sequences

To survey the diversity of anuran species in Bangladesh, we compared mitochondrial 16S rRNA gene sequences (approximately 1.4 kbp) from 107 Bangladesh frog specimens. The results of genetic divergence and phylogenetic analyses incorporating data from related species revealed the occurrence of at least eight cryptic species. Hoplobatrachus tigerinus from two districts diverged considerably, indicating the involvement of a cryptic species. Two Fejervarya sp. (large and medium types) and Hylarana cf. taipehensis formed lineages distinct from related species and are probably new species. Microhyla cf. ornata differed from M. ornata with respect to type locality area and involved two distinct species. In addition, we found that Hylarana sp. and Microhyla sp. did not match congeners examined to date in either morphology or 16S rRNA sequence. The occurrence of M. fissipes was tentatively suggested. Consequently, at least, 19 species were found from Bangladesh in this study. These findings revealed a rich anuran biodiversity in Bangladesh, which is unexpected considering the rather simple topographic features of the country.     

Genetic diversity and spatial genetic structure support the specialist-generalist variation hypothesis in two sympatric woodpecker species

Species are often arranged along a continuum from “specialists” to “generalists”. Specialists typically use fewer resources, occur in more patchily distributed habitats and have overall smaller population sizes than generalists. Accordingly, the specialist-generalist variation hypothesis (SGVH) proposes that populations of habitat specialists have lower genetic diversity and are genetically more differentiated due to reduced gene flow compared to populations of generalists. Here, expectations of the SGVH were tested by examining genetic diversity, spatial genetic structure and contemporary gene flow in two sympatric woodpecker species differing in habitat specialization. Compared to the generalist great spotted woodpecker (Dendrocopos major), lower genetic diversity was found in the specialist middle spotted woodpecker (Dendrocoptes medius). Evidence for recent bottlenecks was revealed in some populations of the middle spotted woodpecker, but in none of the great spotted woodpecker. Substantial spatial genetic structure and a significant correlation between genetic and geographic distances were found in the middle spotted woodpecker, but only weak spatial genetic structure and no significant correlation between genetic and geographic distances in the great spotted woodpecker. Finally, estimated levels of contemporary gene flow did not differ between the two species. Results are consistent with all but one expectations of the SGVH. This study adds to the relatively few investigations addressing the SGVH in terrestrial vertebrates.

     

Cryptic diversity in the mtDNA of the ornamental fish Carnegiella strigata

Mitochondrial DNA ( mtDNA) sequences of the marbled hatchetfish Carnegiella strigata, an ornamental fish exported from the Negro River, was examined to determine its genetic diversity and population structure in blackwater rivers (Negro and Uatumã Rivers) in the central Amazon. Analyses of a 646 bp fragment of the ATPase 6/8 mtDNA gene revealed two monophyletic lineages of C. strigata with considerable genetic distance between them (10–12%), suggesting that these lineages should not be considered a single stock. Furthermore, there were strong differences in the geographical distribution of the lineages. These results indicate a past association between drainages of the Negro and Uatumã Rivers. They also suggest that, in the Negro River, its main tributary, the Branco River, may act as a geographical barrier and potentially an ecological barrier between populations of the middle and lower portions of the river.     

Vertical distribution behaviour and its spatial variation in late-stage larvae of coral-reef fishes during the day

Daytime vertical distribution behaviour of settlement-stage reef-fish larvae in the upper 18?m was documented by diver observations of 497 pelagic larvae of 7 species 100–1000?m offshore of Lizard Island, Great Barrier Reef. Four species were studied on 2 sides of the island. Depth amplitude and depth frequency differed among species, locations and times. Four pomacentrids had modal depths in the upper 30–50% of the water column. A lutjanid and 2 chaetodontids had modal depths 0–2?m deeper than the deepest-swimming pomacentrid. On the leeward side, 6 of 7 species swam deeper and/or more variably offshore. On the windward side, 1 of 4 species swam deeper or more variably offshore. No larvae swam deeper than 18?m on the leeward side, but 31% of larvae of 3 species did so on the windward side. Three of 4 species swam deeper and/or more variably on the windward than leeward side. Vertical distributions in relatively shallow water are apparently strongly influenced by water-column depth and bottom type.     

Microbial diversity in bighorn sheep revealed by culture-independent methods

We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.     

Simultaneous analysis of five molecular markers provides a well-supported phylogenetic hypothesis for the living bony-tongue fishes (Osteoglossomorpha: Teleostei)

Fishes of the Superorder Osteoglossomorpha (the "bonytongues") constitute a morphologically heterogeneous group of basal teleosts, including highly derived subgroups such as African electric fishes, the African butterfly fish, and Old World knifefishes. Lack of consensus among hypotheses of osteoglossomorph relationships advanced during the past 30 years may be due in part to the difficulty of identifying shared derived characters among the morphologically differentiated extant families of this group. In this study, we present a novel phylogenetic hypothesis for this group, based on the analysis of more than 4000 characters from five molecular markers (the mitochondrial cytochrome b, 12S and 16S rRNA genes, and the nuclear genes RAG2 and MLL). Our taxonomic sampling includes one representative of each extant non-mormyrid osteoglossomorph genus, one representative for the monophyletic family Mormyridae, and four outgroup taxa within the basal Teleostei. Maximum parsimony analysis of combined and equally weighted characters from the five molecular markers and Bayesian analysis provide a single, well-supported, hypothesis of osteoglossomorph interrelationships and show the group to be monophyletic. The tree topology is the following: (Hiodon alosoides, (Pantodon buchholzi, (((Osteoglossum bicirrhosum, Scleropages sp.), (Arapaima gigas, Heterotis niloticus)), ((Gymnarchus niloticus, Ivindomyrus opdenboschi), ((Notopterus notopterus, Chitala ornata), (Xenomystus nigri, Papyrocranus afer)))))). We compare our results with previously published phylogenetic hypotheses based on morpho-anatomical data. Additionally, we explore the consequences of the long terminal branch length for the taxon Pantodon buchholzi in our phylogenetic reconstruction and we use the obtained phylogenetic tree to reconstruct the evolutionary history of electroreception in the Notopteroidei.