Challenges in the conservation and sustainable use of genetic resources

The meeting on 'Genetic Resources in the Face of New Environmental, Economic and Social Challenges' held in Montpellier (France) from 20-22 September 2011 brought together about 200 participants active in research and management of the genetic diversity of plant, animal, fungal and microbial species. Attendees had the rare opportunity to hear about agronomy, botany, microbiology, mycology, the social sciences and zoology in the same conference. The research teams presented the results of about 50 projects funded by the French Foundation for Research on Biodiversity to preserve genetic diversity carried out in Africa, Asia, Europe and the Americas. These projects aimed to better understand and manage genetic resources in a rapidly changing world (e.g. structural changes in the agricultural industry, the need for climate change mitigation and adaptation, the challenge of achieving food security despite the growing world population and changing dietary habits, the opportunities provided by the many new molecular biology tools, the problems caused by widespread scientific budget cuts). The meeting also hosted some roundtables open to all participants which provided a forum to establish a much needed dialogue between policy-makers, managers and researchers.     

Marine incursion into East Asia: a forgotten driving force of biodiversity

Episodic marine incursion has been a major driving force in the formation of present-day diversity. Marine incursion is considered to be one of the most productive ‘species pumps’ particularly because of its division and coalescence effects. Marine incursion events and their impacts on diversity are well documented from South America, North America and Africa; however, their history and impacts in continental East Asia largely remain unknown. Here, we propose a marine incursion scenario occurring in East Asia during the Miocene epoch, 10–17 Ma. Our molecular phylogenetic analysis of Platorchestia talitrids revealed that continental terrestrial populations (Platorchestia japonica) form a monophyletic group that is the sister group to the Northwest Pacific coastal species Platorchestia pacifica. The divergence time between the two species coincides with Middle Miocene high global sea levels. We suggest that the inland form arose as a consequence of a marine incursion event. This is the first solid case documenting the impact of marine incursion on extant biodiversity in continental East Asia. We believe that such incursion event has had major impacts on other organisms and has played an important role in the formation of biodiversity patterns in the region.     

Sustainable long-term conservation of rare cattle breeds using rotational AI sires

The development of inbreeding in rotation breeding schemes, sequentially using artificial insemination (AI) sires over generations, was investigated for a full AI scheme. Asymptotic prediction formulae of inbreeding coefficients were established when the first rotation list of AI sires (possibly related) was in use. Simulated annealing provided the optimal rotation order of sires within this list, when the sires were related. These methods were also used for subsequent rotation lists, needed by the exhaustion of semen stores for the first bulls. Simulation was carried out starting with groups of independent sires, with different sizes. To generate a yearly inbreeding rate substantially lower than 0.05% (considered to be within reach by conventional conservation schemes using frequent replacements), the results obtained showed that the number of sires should be at least 10–15 and that the same sires should be used during at least 50 years. The ultimate objective was to examine the relevance of implementing rotation in breeding schemes on the actual rare French cattle breeds under conservation. The best candidate for such a test was the Villard-de-Lans breed (27 bulls and 73 000 doses for only 340 females) and it turned out to be the best performer with an inbreeding coefficient of only 7.4% after 500 years and five different sire lists. Due to the strong requirements on semen stores and on the stability of population size, actual implementation of this kind of conservation scheme was recommended only in special (''niche'') cattle populations.     

A spectacular new Philippine monitor lizard reveals a hidden biogeographic boundary and a novel flagship species for conservation

As humans continue to explore the last uncharted regions of the planet, discoveries of previously unknown species of large vertebrates have become infrequent. Here, we report on the discovery of a spectacular new species of giant, secretive, frugivorous, forest monitor lizard (Genus: Varanus) from the forests of the northern Philippines. Using data from morphology and mitochondrial and nuclear DNA sequences, we demonstrate the taxonomic distinctiveness of this new 2 m long species and provide insight into its historical biogeography and systematic affinities. Our molecular phylogenetic analyses indicate that the new species is closely related to Varanus olivaceus (from southern Luzon and nearby islands), but it differs from this and other varanids with respect to characteristics of scalation, colour pattern, body size, anatomy of the reproductive organs and genetic divergence. The new species appears to be restricted to forests of the central and northern Sierra Madre mountain range; it is separated from the range of V. olivaceus by a more than 150 km stretch that includes at least three low-elevation river valley barriers to dispersal. This discovery identifies a seldom-perceived biogeographic boundary and emphasizes the need for continued biodiversity research in the megadiverse conservation hotspot of the Philippines. It is anticipated that the new species will serve as an important flagship species for conservation efforts aimed at preserving the remaining forests of northern Luzon.     

Prevalence of Haplorchis taichui in field-collected snails: a molecular approach

The prevalence of the cercarial stage of an intestinal trematode, Haplorchis taichui, in thiarid snails (Gastropoda: Thiaridae) was investigated using light microscope and species-specific PCR procedures. A total of 988 snails were collected from Mae Taeng district, Chiang Mai province, northern Thailand, which comprised of 3 species; Melanoides tuberculata, Tarebia granifera, and Thiara scabra. The overall prevalence of pleurolophocercous cercariae was 21.7% as determined by the morphology. For genetic detection of H. taichui infection in snails, 2 primers Hapt_F (5'-GGCCAACGCAATCGTCATCC-3') and Hapt_R (5'-GCGTCGGGTTTCAGACATGG-3'), were used. The genomic DNA of H. taichui, which was used as a positive control, gave an amplification of the 256 bp fragment. The overall prevalence of H. taichui from specific PCR was 9.7%. The proportion of H. taichui among the pleurolophocercous cercariae in this study was 44.9%.     

Conjugation genes are common throughout the genus Rickettsia and are transmitted horizontally

Rickettsia are endosymbionts of arthropods, some of which are vectored to vertebrates where they cause disease. Recently, it has been found that some Rickettsia strains harbour conjugative plasmids and others encode some conjugative machinery within the bacterial genome. We investigated the distribution of these conjugation genes in a phylogenetically diverse collection of Rickettsia isolated from arthropods. We found that these genes are common throughout the genus and, in stark contrast to other genes in the genome, conjugation genes are frequently horizontally transmitted between strains. There is no evidence to suggest that these genes are preferentially transferred between phylogenetically related strains, which is surprising given that closely related strains infect similar host species. In addition to detecting patterns of horizontal transmission between diverse Rickettsia species, these findings have implications for the evolution of pathogenicity, the evolution of Rickettsia genomes and the genetic manipulation of intracellular bacteria.     

Flying shells: historical dispersal of marine snails across Central America

The geological rise of the Central American Isthmus separated the Pacific and the Atlantic oceans about 3 Ma, creating a formidable barrier to dispersal for marine species. However, similar to Simpson's proposal that terrestrial species can 'win sweepstakes routes'-whereby highly improbable dispersal events result in colonization across geographical barriers-marine species may also breach land barriers given enough time. To test this hypothesis, we asked whether intertidal marine snails have crossed Central America to successfully establish in new ocean basins. We used a mitochondrial DNA genetic comparison of sister snails (Cerithideopsis spp.) separated by the rise of the Isthmus. Genetic variation in these snails revealed evidence of at least two successful dispersal events between the Pacific and the Atlantic after the final closure of the Isthmus. A combination of ancestral area analyses and molecular dating techniques indicated that dispersal from the Pacific to the Atlantic occurred about 750 000 years ago and that dispersal in the opposite direction occurred about 72 000 years ago. The geographical distribution of haplotypes and published field evidence further suggest that migratory shorebirds transported the snails across Central America at the Isthmus of Tehuantepec in southern Mexico. Migratory birds could disperse other intertidal invertebrates this way, suggesting the Central American Isthmus may not be as impassable for marine species as previously assumed.     

Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony,likelihood and species tree inference methods

Background and aims

Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree–species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera.

Methods

DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree–species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted.

Key Results

Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia–Lepidozamia–Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia.

Conclusions

A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.     

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l⁻¹ each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l⁻¹ each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.     

Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China

Background and Aims

The genus Allium comprises more than 800 species, placing it among the largest monocotyledonous genera. It is a variable group that is spread widely across the Holarctic region. Previous studies of Allium have been useful in identifying and assessing its evolutionary lineages. However, there are still many gaps in our knowledge of infrageneric taxonomy and evolution of Allium. Further understanding of its phylogeny and biogeography will be achieved only through continued phylogenetic studies, especially of those species endemic to China that have often been excluded from previous analyses. Earlier molecular studies have shown that Chinese Allium is not monophyletic, so the goal of the present study was to infer the phylogeny and biogeography of Allium and to provide a classification of Chinese Allium by placement of Chinese species in the context of the entire phylogeny.

Methods

Phylogenetic studies were based on sequence data of the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast rps16 intron, analysed using parsimony and Bayesian approaches. Biogeographical patterns were conducted using statistical dispersal–vicariance analysis (S-DIVA).

Key Results

Phylogenetic analyses indicate that Allium is monophyletic and consists of three major clades. Optimal reconstructions have favoured the ancestors of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum as originating in eastern Asia.

Conclusions

Phylogenetic analyses reveal that Allium is monophyletic but that some subgenera are not. The large genetic distances imply that Allium is of ancient origin. Molecular data suggest that its evolution proceeded along three separate evolutionary lines. S-DIVA indicates that the ancestor of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum originated from eastern Asia and underwent different biogeographical pathways. A taxonomic synopsis of Chinese Allium at sectional level is given, which divides Chinese Allium into 13 subgenera and 34 sections.     

The phylogenetic affinities of Crossley's babbler (Mystacornis crossleyi): adding a new niche to the vanga radiation of Madagascar

Crossley's babbler (Mystacornis crossleyi) is a passerine endemic to Madagascar. Traditionally, it has been classified as a babbler (Timaliidae), although affinities with warblers and vangas have been suggested. We investigated the phylogenetic affinities of Crossley's babbler using sequence data from two nuclear introns (myoglobin intron 2 and beta-fibrinogen intron 5) and one mitochondrial gene (ND2). We present for the first time (to our knowledge) a molecular phylogeny that confidently places this enigmatic species within the vangas (Vangidae). The inclusion of Crossley's babbler within the vangas adds another foraging niche--gleaning small invertebrates from the ground-to this already large adaptive radiation of songbirds.     

Complete Mitochondrial Genome of Haplorchis taichui and Comparative Analysis with Other Trematodes

Mitochondrial genomes have been extensively studied for phylogenetic purposes and to investigate intra- and interspecific genetic variations. In recent years, numerous groups have undertaken sequencing of platyhelminth mitochondrial genomes. Haplorchis taichui (family Heterophyidae) is a trematode that infects humans and animals mainly in Asia, including the Mekong River basin. We sequenced and determined the organization of the complete mitochondrial genome of H. taichui. The mitochondrial genome is 15,130 bp long, containing 12 protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). Like other trematodes, it does not encode the atp8 gene. All genes are transcribed from the same strand. The ATG initiation codon is used for 9 protein-coding genes, and GTG for the remaining 3 (nad1, nad4, and nad5). The mitochondrial genome of H. taichui has a single long non-coding region between trnE and trnG. H. taichui has evolved as being more closely related to Opisthorchiidae than other trematode groups with maximal support in the phylogenetic analysis. Our results could provide a resource for the comparative mitochondrial genome analysis of trematodes, and may yield genetic markers for molecular epidemiological investigations into intestinal flukes.     

Extreme convergence in stick insect evolution: phylogenetic placement of the Lord Howe Island tree lobster

The ‘tree lobsters’ are an enigmatic group of robust, ground-dwelling stick insects (order Phasmatodea) from the subfamily Eurycanthinae, distributed in New Guinea, New Caledonia and associated islands. Its most famous member is the Lord Howe Island stick insect Dryococelus australis (Montrouzier), which was believed to have become extinct but was rediscovered in 2001 and is considered to be one of the rarest insects in the world. To resolve the evolutionary position of Dryococelus, we constructed a phylogeny from approximately 2.4 kb of mitochondrial and nuclear sequence data from representatives of all major phasmatodean lineages. Our data placed Dryococelus and the New Caledonian tree lobsters outside the New Guinean Eurycanthinae as members of an unrelated Australasian stick insect clade, the Lanceocercata. These results suggest a convergent origin of the ‘tree lobster’ body form. Our reanalysis of tree lobster characters provides additional support for our hypothesis of convergent evolution. We conclude that the phenotypic traits leading to the traditional classification are convergent adaptations to ground-living behaviour. Our molecular dating analyses indicate an ancient divergence (more than 22 Myr ago) between Dryococelus and its Australian relatives. Hence, Dryococelus represents a long-standing separate evolutionary lineage within the stick insects and must be regarded as a key taxon to protect with respect to phasmatodean diversity.     

Molecular and morphological analysis of the critically endangered Fijian iguanas reveals cryptic diversity and a complex biogeographic history

The Pacific iguanas of the Fijian and Tongan archipelagos are a biogeographic enigma in that their closest relatives are found only in the New World. They currently comprise two genera and four species of extinct and extant taxa. The two extant species, Brachylophus fasciatus from Fiji, Tonga, and Vanuatu and Brachylophus vitiensis from western Fiji, are of considerable conservation concern with B. vitiensis listed as critically endangered. A recent molecular study has shown that Brachylophus comprised three evolutionarily significant units. To test these conclusions and to reevaluate the phylogenetic and biogeographic relationships within Brachylophus, we generated an mtDNA dataset consisting of 1462 base pairs for 61 individuals from 13 islands, representing both Brachylophus species. Unweighted parsimony analyses and Bayesian analyses produced a well-resolved phylogenetic hypothesis supported by high bootstrap values and posterior probabilities within Brachylophus. Our data reject the monophyly of specimens previously believed to comprise B. fasciatus. Instead, our data demonstrate that living Brachylophus comprise three robust and well-supported clades that do not correspond to current taxonomy. One of these clades comprises B. fasciatus from the Lau group of Fiji and Tonga (type locality for B. fasciatus), while a second comprises putative B. fasciatus from the central regions of Fiji, which we refer to here as B. n. sp. Animals in this clade form the sister group to B. vitiensis rather than other B. fasciatus. We herein describe this clade as a new species of Brachylophus based on molecular and morphological data. With only one exception, every island is home to one or more unique haplotypes. We discuss alternative biogeographic hypotheses to explain their distribution in the Pacific and the difficulties of distinguishing these. Together, our molecular and taxonomic results have important implications for future conservation initiatives for the Pacific iguanas.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened species.     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

Identification of a Secreted Fatty Acid and Retinol-Binding Protein (Hp-FAR-1) from Heligmosomoides polygyrus

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L(4)) H. polygyrus cDNA expression library using antibodies raised against L(4) stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.     

Thaptomys Thomas 1915 (Rodentia, Sigmodontinae, Akodontini) with karyotypes 2n = 50, FN = 48, and 2n = 52, FN = 52: Two monophyletic lineages recovered by molecular phylogeny

A novel karyotype with 2n = 50, FN = 48, was described for specimens of Thaptomys collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita, 2n = 52, FN = 52, found in other localities. It was hence proposed that the 2n = 50 karyotype could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosomal rearrangements observed, along with the geographic distance, might represent a reproductive barrier between both forms. Phylogenetic analyses using maximum parsimony and maximum likelihood based on partial cytochrome b sequences with 1077 bp were performed, attempting to establish the relationships among the individuals with distinct karyotypes along the geographic distribution of the genus; the sample comprised 18 karyotyped specimens of Thaptomys, encompassing 15 haplotypes, from eight different localities of the Atlantic Rainforest. The intra-generic relationships corroborated the distinct diploid numbers, once both phylogenetic reconstructions recovered two monophyletic lineages, a northeastern clade grouping the 2n = 50 and a southeastern clade with three subclades, grouping the 2n = 52 karyotype. The sequence divergence observed between their individuals ranged from 1.9% to 3.5%.     

Genome Similarity Implies that Citrus-Parasitic Burrowing Nematodes do not Represent a Unique Species

Burrowing nematodes from Central America, Dominican Republic, Florida, Guadeloupe, Hawaii, and Puerto Rico were characterized for their ability to parasitize citrus, but citrus parasites were found only in Florida. Sequence tag sites originally amplified from a citrus-parasitic burrowing nematode were polymorphic among 37 burrowing nematode isolates and were not correlated with citrus parasitism, nematode isolate collection site, or amplification of a 2.4-kb sequence tag site (DK#1). Results of a RAPD analysis and characterization of the isozymes phosphoglucose isomerase, lactate dehydrogenase, and malate dehydrogenase indicated that the burrowing nematode isolates were highly similar. Citrus parasitism in Florida appears to be associated with limited changes in the burrowing nematode genome. Findings did not substantiate a previous report that R. citrophilus was present in Hawaii. Overall, these data do not support assignment of sibling species status to burrowing nematodes that differ with respect to citrus parasitism.