Molecular Ecology Resources

Galindo, L. A.; Puillandre, N.; Strong, E. E.; Bouchet, P.

doi: 10.1111/1755-0998.12231pmid: 24450943

Extracting DNA from gastropods presents particular difficulties due to the capacity of the living animal to retract into the shell, resulting in poor penetration of the ethanol into the tissues. Because the shell is essential to establish the link between sequences and traditional taxonomic identity, cracking the shell to facilitate fixation is not ideal. Several methods are currently in routine use to overcome this difficulty, including chemical relaxation, drilling the shell and boiling. Most of these methods are time‐consuming, may be safety hazards and constitute a bottleneck in the preparation of large numbers of specimens in the field. We have experimented with a method traditionally used to clean shells that involves placing the living gastropods in a microwave (MW) oven; the electromagnetic radiation very quickly heats both the animal and the water trapped inside the shell, resulting in separation of the muscles that anchor the animal to the shell. Done properly, the body can be removed intact from the shell and the shell voucher is preserved undamaged. To test the method, the bodies of live‐collected specimens from two gastropod species were separated from their shell by microwaving and by anesthetizing/drilling. After identical extraction and PCR procedures, the gels showed no difference in DNA quantity or quality, and the resulting sequences are identical within species. The method was then implemented on a large scale during expeditions, resulting in higher percentage of DNA extraction success. The MWs are also effective for quickly and easily removing other molluscs from their shells, that is, bivalves and scaphopods. Workflows implementing the MW technique show a three‐ to fivefold increase in productivity compared with other methods.

Luximon, N.; Petit, E. J.; Broquet, T.

doi: 10.1111/1755-0998.12224pmid: 24400787

Models of isolation‐by‐distance formalize the effects of genetic drift and gene flow in a spatial context where gene dispersal is spatially limited. These models have been used to show that, at an appropriate spatial scale, dispersal parameters can be inferred from the regression of genetic differentiation against geographic distance between sampling locations. This approach is compelling because it is relatively simple and robust and has rather low sampling requirements. In continuous populations, dispersal can be inferred from isolation‐by‐distance patterns using either individuals or groups as sampling units. Intrigued by empirical findings where individual samples seemed to provide more power, we used simulations to compare the performances of the two methods in a range of situations with different dispersal distributions. We found that sampling individuals provide more power in a range of dispersal conditions that is narrow but fits many realistic situations. These situations were characterized not only by the general steepness of isolation‐by‐distance but also by the intrinsic shape of the dispersal kernel. The performances of the two approaches are otherwise similar, suggesting that the choice of a sampling unit is globally less important than other settings such as a study's spatial scale.

Pinosio, S.; González‐Martínez, S. C.; Bagnoli, F.; Cattonaro, F.; Grivet, D.; Marroni, F.; Lorenzo, Z.; Pausas, J. G.; Verdú, M.; Vendramin, G. G.

doi: 10.1111/1755-0998.12232pmid: 24450970

Waud, Michael; Busschaert, Pieter; Ruyters, Stefan; Jacquemyn, Hans; Lievens, Bart

doi: 10.1111/1755-0998.12229pmid: 24460947

Although the number of studies investigating mycorrhizal associations in orchids has increased in recent years, the fungal communities associating with orchids and how they differ between species and sites remain unclear. Recent research has indicated that individual orchid plants may associate with several fungi concurrently, implying that to study mycorrhizal associations in orchids the fungal community should be assessed, rather than the presence of individual dominant fungal species or strains. High‐throughput sequencing methods, such as 454 pyrosequencing, are increasingly used as the primary tool for such analyses. However, many studies combine universal primers from previous phylogenetic or ecological studies to generate amplicons suitable for 454 pyrosequencing without first critically evaluating their performance, potentially resulting in biased fungal community descriptions. Here, following in silico primer analysis we evaluated the performance of different combinations of existing PCR primers to characterize orchid mycorrhizal communities using 454 pyrosequencing by analysis of both an artificially assembled community of mycorrhizal fungi isolated from diverse orchid species and root samples from three different orchid species (Anacamptis morio, Ophrys tenthredinifera and Serapias lingua). Our results indicate that primer pairs ITS3/ITS4OF and ITS86F/ITS4, targeting the internal transcribed spacer‐2 (ITS‐2) region, outperformed other tested primer pairs in terms of number of reads, number of operational taxonomic units recovered from the artificial community and number of different orchid mycorrhizal associating families detected in the orchid samples. Additionally, we show the complementary specificity of both primer pairs, making them highly suitable for tandem use when studying the diversity of orchid mycorrhizal communities.

, ; Chen, Hongjian; Lin, Gonghua; Ma, Junying; Su, Jianping; Wang, Zuyun; Whiting, Michael F.; Zhang, Tongzuo; Zhao, Fang

doi: 10.1111/1755-0998.12263pmid: 24751167

This article documents the public availability of transcriptome sequence data and assembled, annotated unigenes for the marmot flea, Oropsylla silantiewi.1

Kenkel, C. D.; Sheridan, C.; Leal, M. C.; Bhagooli, R.; Castillo, K. D.; Kurata, N.; McGinty, E.; Goulet, T. L.; Matz, M. V.

doi: 10.1111/1755-0998.12218pmid: 24354729

Gene expression biomarkers can enable rapid assessment of physiological conditions in situ, providing a valuable tool for reef managers interested in linking organism physiology with large‐scale climatic conditions. Here, we assessed the ability of quantitative PCR (qPCR)‐based gene expression biomarkers to evaluate (i) the immediate cellular stress response (CSR) of Porites astreoides to incremental thermal stress and (ii) the magnitude of CSR and cellular homeostasis response (CHR) during a natural bleaching event. Expression levels largely scaled with treatment temperature, with the strongest responses occurring in heat‐shock proteins. This is the first demonstration of a ‘tiered’ CSR in a coral, where the magnitude of expression change is proportional to stress intensity. Analysis of a natural bleaching event revealed no signature of an acute CSR in normal or bleached corals, indicating that the bleaching stressor(s) had abated by the day of sampling. Another long‐term stress CHR‐based indicator assay was significantly elevated in bleached corals, although assay values overall were low, suggesting good prospects for recovery. This study represents the first step in linking variation in gene expression biomarkers to stress tolerance and bleaching thresholds in situ by quantifying the severity of ongoing thermal stress and its accumulated long‐term impacts.

Meirmans, Patrick G.

doi: 10.1111/1755-0998.12216pmid: 24373147

The estimation of migration rates using molecular markers is an important aspect of many population genetic studies. Several different methods are available for estimating migration, but most of these make multiple limiting assumptions. One method that is relatively free from assumptions is bayesass, which uses assignment methods in a Bayesian framework. However, when tested using simulated data, this method was found to have problems with the convergence of the Markov chain Monte Carlo. Here, I perform a literature study to test whether these convergence problems are also present when bayesass is used to estimate migration rates from empirical data. A review of 100 studies that have used bayesass shows that this is indeed the case. The estimated proportions of nonmigrants were mostly either close to 2/3 or 1, indicating that the MCMC tends to get trapped near the bounds of the prior distribution. In addition, I found that the quality of the inference was negatively affected by the number of sampled populations, but increased with increasing numbers of sampled individuals and with the strength of the population structure as measured by FST. Based on these results, I give several recommendations that should help to reduce problems when using bayesass with empirical data. Most importantly, I argue that researchers should be more realistic about inferences of migration rates and that bayesass will give optimal results only when the experiment has been especially designed around its use.

Ilves, Katriina L.; López‐Fernández, Hernán

doi: 10.1111/1755-0998.12222pmid: 24410873

Cichlid fishes (family Cichlidae) are models for evolutionary and ecological research. Massively parallel sequencing approaches have been successfully applied to study relatively recent diversification in groups of African and Neotropical cichlids, but such technologies have yet to be used for addressing larger‐scale phylogenetic questions of cichlid evolution. Here, we describe a process for identifying putative single‐copy exons from five African cichlid genomes and sequence the targeted exons for a range of divergent (>tens of millions of years) taxa with probes designed from a single reference species (Oreochromis niloticus, Nile tilapia). Targeted sequencing of 923 exons across 10 cichlid species that represent the family's major lineages and geographic distribution resulted in a complete taxon matrix of 564 exons (649 549 bp), representing 559 genes. Maximum likelihood and Bayesian analyses in both species tree and concatenation frameworks yielded the same fully resolved and highly supported topology, which matched the expected backbone phylogeny of the major cichlid lineages. This work adds to the body of evidence that it is possible to use a relatively divergent reference genome for exon target design and successful capture across a broad phylogenetic range of species. Furthermore, our results show that the use of a third‐party laboratory coupled with accessible bioinformatics tools makes such phylogenomics projects feasible for research groups that lack direct access to genomic facilities. We expect that these resources will be used in further cichlid evolution studies and hope the protocols and identified targets will also be useful for phylogenetic studies of a wider range of organisms.

Lapègue, S.; Harrang, E.; Heurtebise, S.; Flahauw, E.; Donnadieu, C.; Gayral, P.; Ballenghien, M.; Genestout, L.; Barbotte, L.; Mahla, R.; Haffray, P.; Klopp, C.

doi: 10.1111/1755-0998.12230pmid:

Salas‐Leiva, Dayana E.; Meerow, Alan W.; Francisco‐Ortega, Javier; Calonje, Michael; Griffith, M. Patrick; Stevenson, Dennis W.; Nakamura, Kyoko

doi: 10.1111/1755-0998.12228pmid: 24444413

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single‐copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: (i) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and (ii) a tblastX search of Arabidopsis‐Populus‐Vitis‐Oryza Shared Single‐Copy genes (APVO SSC) against the EST Zamia databases in GenBank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases, the matching GenBank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginkgo biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single‐copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Zamia pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.