Molecular species delimitation of a symbiotic fig-pollinating wasp species complex reveals extreme deviation from reciprocal partner specificity

Background: Symbiotic relationships have contributed to major evolutionary innovations, the maintenance of fundamental ecosystem functions, and the generation and maintenance of biodiversity. However, the exact nature of host/symbiont associations, which has important consequences for their dynamics, is often poorly known due to limited understanding of symbiont taxonomy and species diversity. Among classical symbioses, figs and their pollinating wasps constitute a highly diverse keystone resource in tropical forest and savannah environments. Historically, they were considered to exemplify extreme reciprocal partner specificity (one-to-one host-symbiont species relationships), but recent work has revealed several more complex cases. However, there is a striking lack of studies with the specific aims of assessing symbiont diversity and how this varies across the geographic range of the host. Results: Here, we use molecular methods to investigate cryptic diversity in the pollinating wasps of a widespread Australian fig species. Standard barcoding genes and methods were not conclusive, but incorporation of phylogenetic analyses and a recently developed nuclear barcoding gene (ITS2), gave strong support for five pollinator species. Each pollinator species was most common in a different geographic region, emphasising the importance of wide geographic sampling to uncover diversity, and the scope for divergence in coevolutionary trajectories across the host plant range. In addition, most regions had multiple coexisting pollinators, raising the question of how they coexist in apparently similar or identical resource niches. Conclusion: Our study offers a striking example of extreme deviation from reciprocal partner specificity over the full geographical range of a fig-wasp system. It also suggests that superficially identical species may be able to co-exist in a mutualistic setting albeit at different frequencies in relation to their fig host’s range. We show that comprehensive sampling and molecular taxonomic techniques may be required to uncover the true structure of cryptic biodiversity underpinning intimate ecological interactions.

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi. T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. (C) 2010 Elsevier B.V. All rights reserved.

A molecular and morphological investigation of species boundaries and phylogenetic relationships in Australian free-tailed bats Mormopterus (Chiroptera:Molossidae)

The taxonomic uncertainty surrounding several prominent genera of Australian microbat has been a long-standing impediment to research and conservation efforts on these groups. The free-tail bat genus Mormopterus is perhaps the most significant example, with a long history of acknowledged species-level confusion. This study uses a combined molecular and morphological approach to conduct a comprehensive assessment of species and subgeneric boundaries, between-species phylogenetic affinities and within-species phylogeographic structure in Australian members of Mormopterus. Phylogenetic analyses based on 759 base pairs of the NADH Dehydrogenase subunit 2 mitochondrial gene were concordant with species boundaries delineated using an expanded allozyme dataset and by phallic morphology, and also revealed strong phylogeographic structure within two species. The levels of divergence evident in the molecular and morphological analyses led us to recognise three subgenera within Australia: Micronomus, Setirostris subgen. nov. and Ozimops subgen. nov. Within Ozimops we recognise seven Australian species, three of which are new, and none are conspecific with Indo-Papuan species. The family Molossidae now comprises eleven species across three subgenera in Australia, making it the continent's second most speciose family of bats. © CSIRO 2014.

DNA barcoding of Podonomus (Chironomidae, Podonominae) enables stage association of a named species and reveals hidden diversity in Brazilian inselbergs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytogenetic and molecular markers reveal the complexity of the genus Piabina Reinhardt, 1867 (Characiformes: Characidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Can DNA barcoding accurately discriminate megadiverse Neotropical freshwater fish fauna?

Background: The megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region.Results: Of the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence (<2%), application of a complementary character-based nucleotide diagnostic approach proved useful in discriminating them. Additionally, 14 species displayed high intra-specific genetic divergence (>2%), pointing to at least 23 strong candidates for new species.Conclusions: Our study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications. © 2013 Pereira et al.; licensee BioMed Central Ltd.

Identificação molecular de peixes marinhos das regiões Sudeste e Sul do Brasil com ênfase no Estado de São Paulo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytogenetic and DNA barcoding reveals high divergence within the trahira, Hoplias malabaricus (Characiformes: Erythrinidae) from the lower Amazon River

Dados moleculares e citogenéticos tem evidenciado especiação críptica na traíra sul-americana, Hoplias malabaricus. No presente estudo, cariótipos e sequências de DNA barcode de espécimes de sete populações, habitando a região do baixo rio Amazonas, foram analisadas a fim de caracterizar o nível de divergência genética dentro de um único cariomorfo. Todos os espécimes possuem 2n = 40 cromossomos (20m+20sm) os quais são inseridos no grupo de traíras do cariomorfo C. DNA barcode revelou seis haplogrupos, com clara divergência entre populações do Brasil e da Argentina. Os resultados apoiam a hipótese de complexo de espécies e indicam que um único cariomorfo de Hoplias malabaricus pode conter mais de uma espécie.

Genetic identification of bucktooth parrotfish Sparisoma radians (Valenciennes, 1840) (Labridae, Scarinae) by chromosomal and molecular markers

Parrotfishes (Labridae, Scarinae) comprise a large marine fish group of difficult identification, particularly during juvenile phase when the typical morphology and coloration of adults are absent. Therefore, the goal of this study was to test cytogenetic markers and DNA barcoding in the identification of bucktooth parrtotfish Sparisoma radians from the northeastern coast of Brazil. Sequencing of cytochrome c oxidase subunit I (COI) confirmed all studied samples as S. radians, and all showed high similarity (99-100%) with Caribbean populations. The karyotype of this species was divergent from most marine Perciformes, being composed of 2n = 46 chromosomes. These consisted of a large number of metacentric and submetacentric pairs with small amounts of heterochromatin and GC-rich single nucleolar organizer regions (NORs) not syntenic to 5S rDNA clusters. These are the first data about DNA barcoding in parrotfish from the Brazilian province and the first refined chromosomal analysis in Scarinae, providing useful data to a reliable genetic identification of S. radians.

A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes)

DNA barcoding is a recently proposed global standard in taxonomy based on DNA sequences. The two main goals of DNA barcoding methodology are assignment of specimens to a species and discovery of new species. There are two main underlying assumptions: i) reciprocal monophyly of species, and ii) intraspecific divergence is always less than interspecific divergence. Here we present a phylogenetic analysis of the family Potamotrygonidae based on mitochondrial cytochrome c oxidase I gene, sampling 10 out of the 18 to 20 valid species including two non-described species. Potamotrygonidae systematics is still not fully resolved with several still-to-be-described species while some other species are difficult to delimit due to overlap in morphological characters and because of sharing a complex color patterns. Our results suggest that the family passed through a process of rapid speciation and that the species Potamotrygon motoro, P. scobina, and P. orbignyi share haplotypes extensively. Our results suggest that systems of identification of specimens based on DNA sequences, together with morphological and/or ecological characters, can aid taxonomic studies, but delimitation of new species based on threshold values of genetic distances are overly simplistic and misleading.

Sistemática molecular de Utricularia L. (Lentibulariaceae)

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Identificação molecular de espécies da Subfamília Corydoradinae (Siluriformes: Callichthyidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inventário da ictiofauna do alto rio Paraíba do Sul e caracterização molecular das espécies encontradas

The state of São Paulo has four main drainages: Paraná river, Paraíba do Sul river, Ribeira do Iguape river and coastal rivers. The Paraíba do Sul river is born in Sao Paulo and drains an important range of land east of the state. Its ichthyofauna has some similarities and many differences from the continental and coastal drainages which highlights the importance of this study. Surveys conducted in the ichthyofauna of this basin, as in other large river basins in Brazil, is still incomplete. Moreover, there is no consensus about the taxonomic status of many species listed in these surveys. Considering the promising use of DNA barcode as a global system for species identification, the present study is aimed to establishing an inventory of the ichthyofauna of the São Paulo portion of the river Paraíba do Sul and simultaneously build a DNA barcode reference sequence library for fish found. Were obtained and analyzed 354 sequences of the gene cytochrome oxidase c subunit I (COI) belonging to 66 species of São Paulo portion of the Paraíba do Sul river. The average K2P distance between individuals within species of this basin was 0.48%, and 9,87% between species within a genus. Five pairs of species (10 species) showed low levels of interspecific genetic divergence (<2%),but all could be correctly identified. This study showed that the fish species analyzed could be identified efficiently through the use of barcode generating data that can provide information for further studies of this fauna, besides contributing to the global initiative to characterize the species of fish in the world of a molecular point of view. Five pairs of species (10 species) showed low levels of interspecific genetic divergence (<2%), but all could be correctly identified. This study showed that the fish species analyzed could be identified efficiently through the use of barcode generating data that can provide subsidies for further studies in this fauna, as well as ...

Use of anatomical, chemical, and molecular genetic characteristics in the quality control of medicinal species: a case study of sarsaparilla (Smilax spp.)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

DNA Barcoding em Utricularia (Lentibulariaceae)

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV