Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand

Phylogenetic relationships among four species of the Simulium multistriatum group (Diptera: Simuliidae) in Thailand were examined based on two mitochondrial genes (COI, COII) and one nuclear gene (18S/ITS1). Simulium takense was found to be genetically divergent (>20.3% for COI) from the other species, consistent with their distinctive morphology. Simulium chainarongi and S. chaliowae were monophyletic but were included in paraphyletic S. fenestratum. Simulium fenestratum was divided into three distinct lineages with high levels of genetic divergence. This suggests that S. fenestratum is a species complex. Neither morphological nor cytological examinations revealed evidence of sibling species. The clades derived from phylogenetic analyses were found to be correlated with the ecological conditions of larval habitat. Therefore, ecological adaptation may have played a role in black fly diversification and evolution. These results suggest the use of integrated, multidisciplinary approaches for fully understanding black fly biodiversity and systematics.     

Geographical versus ecological isolation of closely related black flies (Diptera: Simuliidae) inferred from phylogeny, geography, and ecology

To investigate patterns of geographical and ecological separation among morphologically similar, closely related species of black flies, we integrated ecological, geographical, and phylogenetic information, based on multiple gene sequences, for 12 species in the subgenus Gomphostilbia in Thailand. Molecular characters supported the monophyly of the Simulium ceylonicum species group, but not of the Simulium batoense species group, suggesting that revisionary work is needed for the latter. Both ecological and geographical isolation of similar taxa were revealed. Stream velocity and altitude were among the principal ecological factors differing between closely related species. Most closely related species in the subgenus Gomphostilbia overlap geographically, suggesting the possibility of sympatric speciation driven by ecological divergence. Geographical isolation via dispersal also might have contributed to species divergence, while Pleistocene climate changes possibly influenced population genetic structure, demographic history, and speciation of some members of the subgenus.     

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host – parasite interactions in black fly mermithids.     

Trichomycete fungi (Zygomycota) associated with the digestive tract of Simulium goeldii Cerqueira & Nunes de Mello and Simulium ulyssesi (Py-Daniel & Coscarón) (Diptera: Simuliidae) larvae,in Central Amazônia,Brazil

Trichomycete fungi are associated with digestive tract of black fly larvae. These fungi have not been studied in Brazil, knowing them and their relationship with black flies could be an alternative to control vector populations. The objectives of this study were to survey the Trichomycete fungi associated with larvae of Simulium goeldii and S. ulyssesi, and to determine if there is specificity and/or difference in the infection rates in these species. Black flies were collected in Central Amaz?nia, Brazil. Three genera of Trichomycetes were found: Harpella, Genistellospora and Smittium. All these fungi were common to both black fly species.     

Genetic panmixia within a narrow contact zone between chromosomally and ecologically distinct black fly sibling species (Diptera: Simuliidae)

Hybrid zones are windows into the speciation process, and their study can give clues into the maintenance and breakdown of species boundaries. Using both genetic and ecological tools, we investigate lineage diversification across a contact zone characterized by chromosome rearrangements. We show that black fly sibling species, Simulium arcticum sensu stricto (s.s.) and Simulium saxosum, lack genetic differentiation at both microsatellite and mtDNA loci in allopatry and sympatry, as well as exhibit high levels of gene flow and continuous chromosome variation in sympatry. Furthermore, hybrid frequencies at the contact zone are similar to those seen between races, rather than species. In contrast, S. arcticum s.s. and S. saxosum maintain ecological differences and distinct habitat associations ‐ the contact zone situated at the margin of suitable habitat for each sibling species. Moreover, gene flow occurs only in a narrow band along an ecological transition. Except for the contact zone, S. arcticum s.s. and S. saxosum hybrids do not occur elsewhere within the sibling species' ranges. Although S. arcticum s.s. and S. saxosum maintain the potential to interbreed freely, we conclude that habitat associations and, perhaps, chromosome systems prevent expansion of ranges and assimilation of lineages.     

Larval description of Simulium (Notolepria) cuasiexiguum and Simulium (Chirostilbia) obesum and new records of black fly species (Diptera: Simuliidae) in the states of São Paulo and Minas Gerais, Brazil

We describe the larval stage of Simulium cuasiexiguum Shelley, Luna Dias, Maia-Herzog & Lowry and Simulium obesum Vulcano and provide new distribution records of four black fly species in the State of S?o Paulo (Simulium cuasiexiguum, Simulium metallicum s.l. Bellardi, Simulium stellatum Gil Azevedo, Figuer6 & Maia-Herzog and Simulium oyapockense s.l. Floch & Abonnenc) and three species in the State of Minas Gerais (Simulium duodenicornium Pepinelli, Hamada & Trivinho-Strixino, Simulium obesum and Lutzsimulium pernigrum Lutz).     

Phenotypic plasticity and genetic isolation-by-distance in the freshwater mussel <Emphasis Type="Italic">Unio pictorum</Emphasis> (Mollusca: Unionoida)

Freshwater mussels (Unionoida) show high intraspecific morphological variability, and some shell morphological traits are believed to be associated with habitat conditions. It is not known whether and which of these ecophenotypic differences reflect underlying genetic differentiation or are the result of phenotypic plasticity. Using 103 amplified fragment length polymorphism (AFLP) markers, we studied population genetics of three paired Unio pictorum populations sampled from two different habitat types (marina and river) along the River Thames. We found genetic differences along the Thames which were consistent with a pattern of isolation by distance and probably reflect limited dispersal via host fish species upon which unionoid larvae are obligate parasites. No consistent genetic differences were found between the two different habitat types suggesting that morphological differences in the degree of shell elongation and the shape of dorso-posterior margin are caused by phenotypic plasticity. Our study provides the first good evidence for phenotypic plasticity of shell shape in a European unionoid and illustrates the need to include genetic data in order properly to interpret geographic patterns of morphological variation.     

Contrasting responses of black fly species (Diptera: Simuliidae) to experimental whole-stream warming

1. As global temperatures continue to rise, assessment of how species within ecological communities respond to shifts in temperature has become increasingly important. However, such assessments require detailed long-term observations or ecosystem-level manipulations that allow for interactions among species and the potential for species dispersal and exchange with the regional species pool.
2. We examined the effects of experimental whole-stream warming on a larval black fly assemblage in southwest Iceland. We used a paired-catchment design, in which we studied the warmed stream and a nearby reference stream for 1 year prior to warming and 2 years during warming and estimated population abundance, biomass, secondary production, and growth rates for larvae of three black fly species.
3. Experimental warming by 3.8°C had contrasting effects on the three black fly species in the assemblage. The abundance, biomass, growth, and production of Prosimulium ursinum decreased in the experimental stream during the warming manipulation. Despite increasing in the reference stream, the abundance, biomass, and production of another species, Simulium vernum, decreased in the experimental stream during warming.
4. In contrast, warming had an overall positive effect on Simulium vittatum. While warming had little effect on the growth of overwintering cohorts of S. vittatum, warming led to an additional cohort during the summer months and increased its abundance, biomass, and production. Overall, family-level production was enhanced by warming, despite variation in species-level responses.
5. Our study illustrates that the effects of climate warming are likely to differ even among closely related species. Moreover, our study highlights the need for further investigation into the uneven effects of warming on individual species and how those variable effects influence food web dynamics and ecosystem function.

     

‘What's larvae got to do with it?’ Disparate patterns of post‐glacial population structure in two benthic marine gastropods with identical dispersal potential

In marine environments, many species have apparently colonized high latitude regions following the last glacial maximum (LGM) yet lack a life-history stage, such as a free-living larva, that is clearly capable of long-distance dispersal. Two hypotheses can explain the modern high latitude distributions of these marine taxa: (1) survival in northern refugia during the LGM or (2) rapid post-glacial dispersal by nonlarval stages. To distinguish these two scenarios, I characterized the genetic structure of two closely related northeastern Pacific gastropods that lack planktonic larvae but which have distributions extending more than 1000 km north of the southern limit of glaciers at the LGM. Despite having identical larval dispersal potential, these closely related species exhibit fundamentally different patterns of genetic structure. In Nucella ostrina, haplotype diversity among northern populations (British Columbia and Alaska) is low, no pattern of isolation by distance exists and a coalescent-based model of population growth indicates that during the LGM population size was reduced to less than 35% of its current size. In the congeneric and often sympatric N. lamellosa, northern populations harbour a diversity of ancient private haplotypes, significant evidence of isolation by distance exists and regional subdivision was found between northern (Alaska) and southern (southern British Columbia, Washington and Oregon) populations. Estimates of coalescent parameters indicate only a modest reduction in population size during the LGM and that northern and southern populations of N. lamellosa split approximately 50 Kyr before the LGM. The patterns are consistent with the hypothesis that N. ostrina recently reinvaded the northeastern Pacific but N. lamellosa survived the LGM in a northern refuge. A comparison of similar studies in this region indicates that depleted levels of genetic variation at high latitudes--evidence suggestive of recent colonization from a southern refuge--is more common among intertidal species that live relatively high on the shore, where exposure times to cold stress in air are longer than for species living lower on the shore. These data suggest that for some faunas, ecological differences between taxa may be more important than larval dispersal potential in determining species' long-term biogeographical responses to climate change.     

First report of a trichomycete fungus (Zygomycota: Trichomycetes) inhabiting larvae of Simulium ochraceum sensu lato Walker (Diptera: Simuliidae) from the Galapagos Islands

A collection of the larval black fly Simulium ochraceum sensu lato Walker was made at six lotic habitats on San Cristobal Island, Galapagos Islands, Ecuador, during May 2003. Sixty-eight larvae were assayed for the presence of gut fungi (Zygomycota: Trichomycetes). A trichomycete, designated as Smittium sp. was found inhabiting the hindgut of 17.6% of S. ochraceum s.l. larvae. Implications of this finding with respect to both host and trichomycete biogeography are discussed.     

Phenotypic divergence but not genetic distance predicts assortative mating among species of a cichlid fish radiation

The hypothesis of ecological divergence giving rise to premating isolation in the face of gene flow is controversial. However, this may be an important mechanism to explain the rapid multiplication of species during adaptive radiation following the colonization of a new environment when geographical barriers to gene flow are largely absent but underutilized niche space is abundant. Using cichlid fish, we tested the prediction of ecological speciation that the strength of premating isolation among species is predicted by phenotypic rather than genetic distance. We conducted mate choice experiments between three closely related, sympatric species of a recent radiation in Lake Mweru (Zambia/DRC) that differ in habitat use and phenotype, and a distantly related population from Lake Bangweulu that resembles one of the species in Lake Mweru. We found significant assortative mating among all closely related, sympatric species that differed phenotypically, but none between the distantly related allopatric populations of more similar phenotype. Phenotypic distance between species was a good predictor of the strength of premating isolation, suggesting that assortative mating can evolve rapidly in association with ecological divergence during adaptive radiation. Our data also reveals that distantly related allopatric populations that have not diverged phenotypically, may hybridize when coming into secondary contact, e.g. upon river capture because of diversion of drainage systems.     

Rapid and repeatable shifts in life‐history timing of Rhagoletis pomonella (Diptera: Tephritidae) following colonization of novel host plants in the Pacific Northwestern United States

Host shifts of phytophagous insect specialists to novel plants can result in divergent ecological adaptation, generating reproductive isolation and potentially new species. Rhagoletis pomonella fruit flies in eastern North America underwent a host shift ~160 ya from native downy hawthorn (Crataegus mollis) to introduced, domesticated apple (Malus domestica). Divergent selection on diapause phenology related to the earlier fruiting time of apples versus downy hawthorns resulted in partial allochronic reproductive isolation between the fly races. Here, we test for how rapid and repeatable shifts in life‐history timing are driving ecological divergence of R. pomonella in the Pacific Northwestern USA. The fly was introduced into the region via larval‐infested apples 40–65 ya and now attacks native black hawthorn (Crataegus douglasii) and introduced ornamental hawthorn (Crataegus monogyna), in addition to early‐ and late‐maturing apple varieties in the region. To investigate the life‐history timing hypothesis, we used a field‐based experiment to characterize the host‐associated eclosion and flight activity patterns of adults, and the feeding times of larvae at a field site in Vancouver, Washington. We also assessed the degree to which differences in host‐fruiting time generate allochronic isolation among apple‐, black hawthorn‐, and ornamental hawthorn‐associated fly populations. We conclude that host‐associated fly populations are temporally offset 24.4% to 92.6% in their seasonal distributions. Our results imply that R. pomonella possesses the capacity for rapid and repeatable shifts in diapause life history to match host‐fruiting phenology, which can generate ecologically based reproductive isolation, and potentially biodiversity in the process.     

Ecological reproductive isolation of coast and inland races of Mimulus guttatus

Adaptive divergence due to habitat differences is thought to play a major role in formation of new species. However it is rarely clear the extent to which individual reproductive isolating barriers related to habitat differentiation contribute to total isolation. Furthermore, it is often difficult to determine the specific environmental variables that drive the evolution of those ecological barriers, and the geographic scale at which habitat-mediated speciation occurs. Here, we address these questions through an analysis of the population structure and reproductive isolation between coastal perennial and inland annual forms of the yellow monkeyflower, Mimulus guttatus. We found substantial morphological and molecular genetic divergence among populations derived from coast and inland habitats. Reciprocal transplant experiments revealed nearly complete reproductive isolation between coast and inland populations mediated by selection against immigrants and flowering time differences, but not postzygotic isolation. Our results suggest that selection against immigrants is a function of adaptations to seasonal drought in inland habitat and to year round soil moisture and salt spray in coastal habitat. We conclude that the coast and inland populations collectively comprise distinct ecological races. Overall, this study suggests that adaptations to widespread habitats can lead to the formation of reproductively isolated species.     

The effect of seston on mortality of Simulium vittatum (Diptera: Simuliidae) from insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis

Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.     

The Simulium vernum group (Diptera: Simuliidae) in Europe: multiple character sets for assessing species status

The value of using characters from multiple sources – chromosomes, ecology, gene sequences, and morphology – to evaluate the species status of closely related black flies is demonstrated for three European members of the Simulium vernum group: Simulium crenobium (Knoz, 1961), Simulium juxtacrenobium Bass & Brockhouse, 1990, and Simulium vernum s.s. Macquart, 1826. Simulium juxtacrenobium is a chromosomally, molecularly, and morphologically distinct species that diverged from S. crenobium and S. vernum s.s. about 2 Mya. It is specialized for intermittent streams, is univoltine, and is recorded for the first time from northern Europe, based on collections from Finland and Sweden, representing a range extension of about 1800 km. In contrast, S. crenobium, although confirmed as a distinct species, differs from S. vernum s.s. by only a few larval and chromosomal characters, and by a breeding habitat restricted to mountain spring brooks. Whereas all four character sets independently support the specific distinctness of S. juxtacrenobium and S. vernum s.s., multiple character sets are required to establish the specific validity of S. crenobium.     

Sympatric serpentine endemic Monardella (Lamiaceae) species maintain habitat differences despite hybridization

Ecological differentiation and genetic isolation are thought to be critical in facilitating coexistence between related species, but the relative importance of these phenomena and the interactions between them are not well understood. Here, we examine divergence in abiotic habitat affinity and the extent of hybridization and introgression between two rare species of Monardella (Lamiaceae) that are both restricted to the same serpentine soil exposure in California. Although broadly sympatric, they are found in microhabitats that differ consistently in soil chemistry, slope, rockiness and vegetation. We identify one active hybrid zone at a site with intermediate soil and above‐ground characteristics, and we document admixture patterns indicative of extensive and asymmetric introgression from one species into the other. We find that genetic distance among heterospecific populations is related to geographic distance, such that the extent of apparent introgression is partly explained by the spatial proximity to the hybrid zone. Our work shows that plant species can maintain morphological and ecological integrity in the face of weak genetic isolation, intermediate habitats can facilitate the establishment of hybrids, and that the degree of apparent introgression a population experiences is related to its geographic location rather than its local habitat characteristics.     

Limited ecological population connectivity suggests low demands on self-recruitment in a tropical inshore marine fish (Eleutheronema tetradactylum: Polynemidae)

The diversity of geographic scales at which marine organisms display genetic variation mirrors the biophysical and ecological complexity of dispersal by pelagic larvae. Yet little is known about the effect of larval ecology on genetic population patterns, partly because detailed data of larval ecology do not yet exist for most taxa. One species for which this data is available is Eleutheronema tetradactylum, a tropical Indo-West Pacific shorefish. Here, we use a partial sequence mitochondrial cytochrome oxidase subunit 1 (COI) marker and five microsatellite loci to survey the genetic structure of E. tetradactylum across northern Australia. Structure was found throughout the range and isolation by distance was strong, explaining approximately 87 and 64% of the genetic variation in microsatellites and mtDNA, respectively. Populations separated by as little as 15 km also showed significant genetic structure, implying that local populations are mainly insular and self-seeding on an ecological time frame. Because the larvae of E. tetradactylum have lower swimming performance and poor orientation compared with other tropical fishes, even modest larval abilities may permit self-recruitment rather than passive dispersal.     

Population structure of the Atlantic sand fiddler crab Uca pugilator along the eastern coast of US revealed by molecular data

The Atlantic sand fiddler crab Uca pugilator is an extremely abundant crab found along the eastern coast of the United States. Fiddler crabs have a life cycle with an obligatory planktonic larval phase of 30-90 days, which might be expected to lead to widespread larval dispersal and consequent genetic homogeneity over considerable distances. However, a large amount of morphological and behavioral variation is found between northern and southern populations along the eastern coast. This study was undertaken to determine the population genetic structure of U.pugilator and to determine whether these differences may have a genetic basis. The population structure of the fiddler crab was analyzed using 472 individuals collected from 12 sites along the eastern coast. PCR-based single stand conformation polymorphism (SSCP) was used to investigate between-site variation in the mitochondrial 16S rRNA gene of these individuals. Analysis of genetic variation indicated frequent gene flow between nearby localities, but much reduced levels between populations separated by larger geographic distances. Thus, despite the potential for high dispersal by planktonic larvae, population differentiation and isolation by distance is evident between northern and southern populations of U.pugilator. A high amount of genetic differentiation (FST=0.3468) was found between northern and southern regions suggesting that the morphological and behavioral differences between these two regions have a genetic basis and may represent subspecies [Current Zoology 55(2):150-157,2009].     

Adaptive divergence vs. environmental plasticity: tracing local genetic adaptation of metamorphosis traits in salamanders

In order to assess the significance of local adaptation relative to environmental plasticity on the evolution of life history traits, we analysed the possible genetic basis of differences between pond- and stream-breeding fire salamanders (Salamandra salamandra) in Germany. These salamanders typically deposit their larvae in small streams, where they grow until they are sufficiently large to metamorphose. However, some populations in Western Germany use ponds as larval habitat. Because habitat quality of streams differs from that of ponds one expects life history differences in the pond animals, which may result either from a plastic response or through genetic differentiation (i.e. local adaptation). Using a phylogeographical analysis of mitochondrial D-loop sequences, we show that both stream and pond populations in Western Germany are derived from a single lineage that recolonized following the last glaciation. This finding suggests that pond breeding originated very recently. Our studies of habitat quality and metamorphic behaviour of larvae in natural ponds and streams disclosed that pond larvae experience a significantly reduced food supply and greater risk of drying than do stream larvae. Pond larvae metamorphose earlier at the cost of reduced mass. Common-environment experiments with pond and stream larvae show that metamorphic behaviour of pond larvae under limited-food conditions is determined genetically and is not simply a plastic response to the differing habitat conditions. These results show that phenotypic plasticity is less important than local adaptation in explaining differences in ecological diversification within this species and suggests the possibility of rapid evolution of genetic adaptations when new habitats are exploited.     

Glacial survival and local adaptation in an alpine leaf beetle

The challenge in defining conservation units so that they represent evolutionary entities has been to combine both genetic properties and ecological significance. Here we make use of the complexity of the European Alps, with their genetic landscape shaped by geographical barriers and postglacial colonization, to examine the correlation between ecological and genetic divergence. Montane species, because of the fragmentation of their present habitat, constitute extreme cases in which to test if genetically distinct subgroups based on neutral markers are also ecologically differentiated and show local adaptation. In the leaf beetle Oreina elongata, populations show variation in host plant use and a patchy distribution throughout the Alps and Apennines. We demonstrate that despite very strong genetic isolation (F(ST) = 0.381), variation in host plant use has led to differences in larval life-history traits between populations only as a secondary effect of host defence chemistry, and not through physiological adaptation to plant nutritional value. We also establish that populations that are more ecologically different in terms of larval performance are also more genetically divergent. In addition, morphological variation used to define subspecies appears to be mirrored in the population genetics of this species, resulting in almost perfect clustering based on microsatellite data. Finally, we argue from their strong genetic structure and congruent distribution that the subspecies of O. elongata were divided among the same glacial refugia within the Alps that have been proposed for alpine plants.