Mol Ecol Research Articles

Development of tools to rapidly identify cryptic species and characterize their genetic diversity in different European kelp species

Marine ecosystems formed by kelp forests are severely threatened by global change and local coastline disturbances in many regions. In order to take appropriate conservation, mitigation, and restoration actions, it is crucial to identify the most diverse populations which could serve as a “reservoir” of genetic diversity. This requires the development of specific tools, such as microsatellite markers to investigate the level and spatial distribution of genetic diversity. Here, we tested new polymorphic microsatellite loci from the genome of the kelp, Laminaria digitata, and tested them for cross-amplification and polymorphism in four closely related congeneric species (Laminaria hyperborea, Laminaria ochroleuca, Laminaria rodriguezii, and Laminaria pallida). Adding these 20 new microsatellite loci to the ten L. digitata loci previously developed by Billot et al. (Mol Ecol 7:1778-1780, 1998) and Brenan et al. (J R Soc Interface 11:1-12, 2014) and to the ten L. ochroleuca loci previously developed by Coelho et al. (Conserv Genet Resource 6:949-950, 2014), we retained a total of 30 polymorphic loci for L. digitata, 21 for L. hyperborea, 16 for L ochroleuca, 18 for L. rodriguezii, and 12 for L. pallida. These markers have been tested for the first time in the last two species. As predicted, the proportion of markers that cross-amplified between species decreased with increasing genetic distance. In addition, as problems of species identification were reported in this family, mainly between L. digitata and Hedophyllum nigripes, but also between L. digitata, L. hyperborea, and L. ochroleuca in areas where their range distributions overlap, we report a rapid PCR identification method based on species-specific COI mitochondrial primers that allows these four species of kelp to be rapidly distinguished.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, has been identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based ‘barcoding gap’, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

Evolution of pathogen response genes associated with increased disease susceptibility during adaptation to an extreme drought in a Brassica rapa plant population

BackgroundPathogens are key components in natural and agricultural plant systems. There is evidence of evolutionary changes in disease susceptibility as a consequence of climate change, but we know little about the underlying genetic basis of this evolution. To address this, we took advantage of a historical seed collection of a Brassica rapa population, which we previously demonstrated evolved an increase in disease susceptibility to a necrotrophic fungal pathogen following a drought.ResultsPreviously, we combined a resurrection experiment with genome-wide sequencing of 124 pooled ancestral and descendant plants. Here, using these previously generated sequence data (Franks et al. in Mol Ecol 25(15):3622–3631, 2016), we show that well-characterized necrotrophic fungal pathogen response (NFPR) genes have evolved, as indicated by changes in allele frequency, between ancestors and descendants, with several of them identified as extreme FST outliers. The jasmonic acid (JA) signaling pathway in particular seems to underlie the evolution of disease susceptibility, in addition to its well characterized role in plastic disease response. We identify a list of 260 genes that are both NFPR genes and are differentially expressed in response to drought, based on publicly available data. We present evidence that five of these genes evolved between ancestors and descendants, suggesting that the drought acted as the evolutionary driver, and that the accompanying increase in disease susceptibility may have been a consequence of genetic pleiotropy.ConclusionsOur study provides evidence that for this population, standing variation in NFPR genes is affected by natural selection related to climate change. Our results reveal potentially important candidates that may underlie trait evolution in both crops and natural systems. Additionally, this trade-off between adaptation to biotic and abiotic stresses is an example of how climate change can have diverse and unexpected consequences.

Can laboratory evolution experiments teach us about natural populations?

The ability to predict how natural populations will evolve and adapt to major changes in environmental conditions has long been of interest to evolutionary biologists and ecologists alike. The reality of global climate change has also created a pressing need for advancement in this particular area of research, as species are increasingly faced with rapid shifts in abiotic and biotic conditions. Evolutionary genomics has the potential to be incredibly useful as we move forward in addressing this need and in particular, evolve and resequence (E&R) studies-where researchers combine experimental evolution with whole-genome sequencing-have an important role to play. However, while E&R studies have shown a great deal of promise in tackling fundamental questions regarding the genetics of adaptation (Long et al., 2015; Schlötterer et al., 2014), it is unclear whether results from laboratory experiments can be directly translated to natural populations. In a From the Cover article in this issue of Molecular Ecology, Hsu et al. (Mol Ecol, 29, 2020) explicitly contend with this issue by examining the overlap between genes implicated in thermal adaptation in a Drosophila melanogaster E&R study and genes identified by comparing natural populations from different latitudinal clines. They report significant correlations between the two sets of temperature-adaptive genes and ultimately conclude that E&R studies can indeed generate insights applicable to populations inhabiting complex natural environments. While more work is needed to assess the generality of these conclusions, Hsu and Belmouaden (Mol Ecol, 29, 2020) contribute an important precedent.

Seabed mining could come at a high price for a unique fauna.

The deep seafloor is teeming with life, most of which remains poorly known to science. It also constitutes an important reserve of natural resources, particularly minerals, that mining companies will start harvesting in the next few years (Nat Rev Earth Environ, 1, 2020, 158). In this context, broad biodiversity assessments of deep-sea ecosystems are urgently needed to establish a baseline prior to mining. However, significant gaps in our taxonomic knowledge and the high cost of sampling in the deep sea limit the effectiveness of conventional morphology-based surveys. In this issue of Molecular Ecology, Laroche et al. (Mol Ecol, 2020) capitalize on high throughput molecular methods to conduct one of the most detailed and rigorous surveys of the composition and biogeography of deep-seafloor metazoan communities to date. The authors show that deep seamounts in the Clarion Clipperton Zone are inhabited by rich metazoan communities that are distinct from those of the surrounding abyssal plains. These results have important conservation implications: if communities on deep seamounts were to persist after large-scale industrial mining operations on the surrounding plains, the seamounts would not serve as appropriate reservoirs to repopulate impacted areas.

Reconstruction of ancient homeobox gene linkages inferred from a new high-quality assembly of the Hong Kong oyster (Magallana hongkongensis) genome

BackgroundHomeobox-containing genes encode crucial transcription factors involved in animal, plant and fungal development, and changes to homeobox genes have been linked to the evolution of novel body plans and morphologies. In animals, some homeobox genes are clustered together in the genome, either as remnants from ancestral genomic arrangements, or due to coordinated gene regulation. Consequently, analyses of homeobox gene organization across animal phylogeny provide important insights into the evolution of genome organization and developmental gene control, and their interaction. However, homeobox gene organization remains to be fully elucidated in several key animal ancestors, including those of molluscs, lophotrochozoans and bilaterians.ResultsHere, we present a high-quality chromosome-level genome assembly of the Hong Kong oyster, Magallana hongkongensis (2n = 20), for which 93.2% of the genomic sequences are contained on 10 pseudomolecules (~ 758 Mb, scaffold N50 = 72.3 Mb). Our genome assembly was scaffolded using Hi-C reads, facilitating a larger scaffold size compared to the recently published M. hongkongensis genome of Peng et al. (Mol Ecol Resources, 2020), which was scaffolded using the Crassostrea gigas assembly. A total of 46,963 predicted gene models (45,308 protein coding genes) were incorporated in our genome, and genome completeness estimated by BUSCO was 94.6%. Homeobox gene linkages were analysed in detail relative to available data for other mollusc lineages.ConclusionsThe analyses performed in this study and the accompanying genome sequence provide important genetic resources for this economically and culturally valuable oyster species, and offer a platform to improve understanding of animal biology and evolution more generally. Transposable element content is comparable to that found in other mollusc species, contrary to the conclusion of another recent analysis. Also, our chromosome-level assembly allows the inference of ancient gene linkages (synteny) for the homeobox-containing genes, even though a number of the homeobox gene clusters, like the Hox/ParaHox clusters, are undergoing dispersal in molluscs such as this oyster.

Genotyping ITS and matK regions of Hedera nepalensis K. Koch in Vietnam

Genotyping ITS and matK regions of Hedera nepalensis K. Koch in Vietnam

Non-invasive genetic identification of two sympatric sister-species: ocelot (Leopardus pardalis) and margay (L. wiedii) in different biomes

The method presented in here allows for the unequivocal differentiation between non-invasive samples of ocelots and margays, when used in combination with the protocol by Roques et al. (Mol Ecol Resour 11:171–175, 2011). It reduced costs and laboratory processing, decreasing the chances of contamination, and therefore facilitating the identification of large numbers of samples by simple PCR amplification, without the need for sequencing. Our protocol was tested on 371 faeces collected in the field in 25 different sites across the distribution areas of both species, and compared to the mtDNA sequencing of 23 of the samples. The concatenation of a commonly used segment of Cytochrome b with other mitochondrial markers such as ATP-8 allows the identification of these two sister-species, but there was not enough resolution when used alone. With this new method, we could identify 98% of the field faeces, obtaining a global rate of 22.3 faeces of ocelot per margay sample in roads and trails located in potential areas of coexistence, suggesting generally lower densities of margay, although margays could be underestimated due to their arboreal habits. In Serra das Almas, located in the Brazilian Caatinga, the number of faeces of ocelot detected was especially elevated. Low densities of ocelots could be favoring higher densities of margays in areas like the Sinaloan dry forests and the Chimalapas montane forests, both in Mexico. Ocelots were found from open to forested habitats, including mosaic cropland and grassland, however margays were only found in forests where average canopy height exceeded 5 m.

SNP Discovery from Single and Multiplex Genome Assemblies of Non-model Organisms.

Population genetic studies of non-model organisms often rely on initial ascertainment of genetic markers from a single individual or a small pool of individuals. This initial screening has been a significant barrier to beginning population studies on non-model organisms (Aitken et al., Mol Ecol 13:1423-1431, 2004; Morin et al., Trends Ecol Evol 19:208-216, 2004). As genomic data become increasingly available for non-model species, SNP ascertainment from across the genome can be performed directly from published genome contigs and short-read archive data. Alternatively, low to medium genome coverage from shotgun NGS library sequencing of single or pooled samples, or from reduced-representation libraries (e.g., capture enrichment; see Ref. "Hancock-Hanser et al., Mol Ecol Resour 13:254-268, 2013") can produce sufficient new data for SNP discovery with limited investment. We describe protocols for assembly of short read data to reference or related species genome contig sequences, followed by SNP discovery and filtering to obtain an optimal set of SNPs for population genotyping using a variety of downstream high-throughput genotyping methods.

Corrigendum.

Molecular EcologyVolume 26, Issue 8 p. 2405-2405 CorrigendumFree Access Corrigendum This article corrects the following: Biogeographic patterns of soil diazotrophic communities across six forests in the North America Qichao Tu, Ye Deng, Qingyun Yan, Lina Shen, Lu Lin, Zhili He, Liyou Wu, Joy D. Van Nostrand, Vanessa Buzzard, Sean T. Michaletz, Brian J. Enquist, Michael D. Weiser, Michael Kaspari, Robert B. Waide, James H. Brown, Jizhong Zhou, Volume 25Issue 12Molecular Ecology pages: 2937-2948 First Published online: May 21, 2016 First published: 17 April 2017 https://doi.org/10.1111/mec.13925AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Qichao Tu, Ye Deng, Qingyun Yan, Lina Shen, Lu Lin, Zhili He, Liyou Wu, Joy D. Van Nostrand, Vanessa Buzzard, Sean T. Michaletz, Brian J. Enquist, Michael D. Weiser, Michael Kaspari, Robert B. Waide, James H. Brown and Jizhong ZhouRegrettably, a grammatical error appeared in the title of the published paper by Tu et al. (2016).The correct title is ‘Biogeographic patterns of soil diazotrophic communities across six forests in North America’. As it was still in review at the time Tu, Q., et al. (2016), Biogeographic patterns of soil diazotrophic communities across six forests in the North America. Mol Ecol, 25: 2937-2948. doi:10.1111/mec.13651 was published, the reference for Zhou et al. (2016) was inadvertently omitted from the list of references. We include it here:Zhou J, Deng Y, Shen L et al. (2016) Temperature mediates continental-scale diversity of microbes in forest soils. Nature Communications. DOI: 10.1038/ncomms12083 Reference Tu Q, Deng Y, Yan Q et al. (2016) Biogeographic patterns of soil diazotrophic communities across six forests in the North America. Molecular Ecology, 25, 2937– 2948. Volume26, Issue8April 2017Pages 2405-2405 ReferencesRelatedInformation

Genetic Differentiation of Colombian Populations of Anopheles darlingi Root (Diptera: Culicidae).

Anopheles darlingi Root is a primary vector of malaria in the neotropic region, a species not just highly anthropophilic but very efficient in transmitting Plasmodium species and considered the most important vector in the Amazon region. The main goal of this study was to determine the genetic structure of the A. darlingi populations using microsatellites (STR) in western and eastern regions of Colombia. DNA extraction was done with the cited protocol of band using the Genomic Prep™ cell and tissue isolation commercial kits. We used the STR reported by Conn et al (Mol Ecol Notes 1: 223-225, 2001). The analysis with STR proved there was a high genetic diversity and significant alterations of the Hardy-Weinberg equilibrium. The greatest genetic diversity was recorded in Mitu (Vaupes) (Na=14, Ho=0.520). The lowest was in Pueblo Nuevo (Cordoba) (Na=12, Ho=0.457). The eastern region and the Mitu (Vaupes) populations presented the highest number of primer alleles (Ap=30; Ap=13; Ap=9), with variations between 0.010 and 0.097. The AMOVA revealed that the whole population underwent moderate genetic differentiation (F ST=0.063, p<0.05). The same differentiation was noticed (0.06<F ST>0.06, p<0.05) with five of the six populations included in this job, and there was a low differentiation in the Las Margaritas (Santander) area (F ST=0.02s3, p<0.05). Our results suggest a slight positive correlation, which does not show a statistical significance between the geographic and genetic distances, probably suggesting that the moderate genetic differentiation found between pairs of populations does not need to be explained for the hypothesis of separation by distance.

Microsatellite Analysis for Identification of Individuals Using Bone from the Extinct Steller's Sea Cow (Hydrodamalis gigas).

Microsatellite DNA can provide more detailed population genetic information than mitochondrial DNA which is normally used to research ancient bone. The methods detailed in this chapter can be utilized for any type of bone. However, for this example, four microsatellite loci were isolated from Steller's sea cow (Hydrodamalis gigas) using published primers for manatee and dugong microsatellites. The primers DduC05 (Broderick et al., Mol Ecol Notes 6:1275-1277, 2007), Tmakb60, TmaSC5 (Pause et al., Mol Ecol Notes 6: 1073-1076, 2007), and TmaE11 (Garcia-Rodriguez et al., Mol Ecol 12:2161-2163, 2000) all successfully amplified microsatellites from H. gigas. The DNA samples were from bone collected on Bering or St. Lawrence Islands. DNA was analyzed using primers with the fluorescent label FAM-6. Sequenced alleles were then used to indicate a difference in the number of repeats and thus a difference in individuals. This is the first time that H. gigas microsatellite loci have been isolated. These techniques for ancient bone microsatellite analysis allow an estimate of population size for a newly discovered St. Lawrence Island sea cow population.

An R version of FPTest for testing differences in allelic count

Allelic count is an important measure of genetic diversity. However, it is a function of sample size and its estimation and interpretation can be biased. Previously, we introduced a random permutation method named FPTest to test the differences in allelic count among oat germplasm groups of variable sample size (Fu et al., Crop Sci 43:1989–1995, 2003) and published a SAS routine for it to facilitate its application (Fu, Mol Ecol Resour 10:398–392, 2010). Here we present an R version of FPTest called FPTestR. This R version considers six types of large marker data and generates four to five output files, making the test more accessible and useful in genetic diversity analysis.

Autosomal and Z-linked microsatellite markers enhanced for cross-species utility and assessed in a range of birds, including species of conservation concern

Microsatellite markers were designed to be of utility for genotyping multiple species of birds, including those of conservation concern, hence saving resources and enabling species/genome comparisons. We used the proven approach of Dawson et al. (Mol Ecol Resour 10:475–494, 2010) and assessed markers in multiple species, including nine species of conservation interest. We ensured both primer sequences matched multiple species (13 loci) or designed primer sets from expressed sequence tags (2 loci). Eleven primer sets were 100 % identical to the zebra finch (Taeniopygia guttata) and a second passerine species and/or the chicken (Gallus gallus). All 15 loci were polymorphic when assessed in a non-source species (Gouldian finch, Erythrura gouldiae) suggesting utility in multiple species. Four of the five Z-linked loci were assessed in at least nine additional species each (including ratites). All were variable in multiple species, demonstrating cross-species utility and potential for identifying Z chromosome rearrangements.

A proposed essential gene discovery pipeline: a Campylobacter jejuni case study.

Genes required for an organism's growth and survival are termed essential and represent potential intervention targets. Following in the footsteps of the genomics era, the "next-gen" genomic era provides vast amounts of genetic information. Sequencing of a representative bacterial pathogen genome has been superseded by sequencing of whole strain collections, whether from environmental or clinical sources (Harris et al., Science 327:469-474, 2010; Lewis et al., J Hosp Infect 75:37-41, 2010; Beres et al., Proc Natl Acad Sci U S A 107:4371-4376, 2010; Qi et al., PLoS Pathog 5:e1000580, 2009; He et al., Proc Natl Acad Sci U S A 107:7527-7532, 2010; Barrick et al., Nature 461:1243-1247, 2009; Sheppard et al., Mol Ecol 22:1051-1064, 2013). However, the challenge of using this information to gain biological insight remains. Nonetheless, this information, in combination with experimental data from the literature, can serve as the framework for gaining a better understanding of an organism's biology. Generic metabolic pathways have long been known, and a number of websites (e.g., KEGG and BioCyc) attempt to map information from genome annotation to metabolic pathways (Kanehisa et al., Nucleic Acids Res 40:D109-D114, 2010; Karp et al., Nucleic Acids Res 33:6083-6089, 2005). Extending this analysis to incorporate metabolic flux models further allows in silico prediction of potential essential genes. Such efforts are of value, either to highlight novel generic antimicrobials or to seek novel treatments for non-paradigm organisms. Such in silico approaches are attractive as they can highlight pathways and genes that would otherwise only be identified by costly and time-consuming laboratory methods.

Does phenology distinguish bitter and sweet African bush mango trees (Irvingia spp., Irvingiaceae)?

This phenological analysis of bitter and sweet bush mango trees is part of their biosystematics. It supports the species distinction hypothesis postulated by Harris (Bull J Bot Nat Belg 65(1–2):143–196, 1996 ) and Lowe et al. (Mol Ecol 9:831–841, 2000 ). African Bush Mango trees are priority food trees in Sub-Saharan Africa. The unclear distinction between bitter and sweet fruited trees is still subject to taxonomic debate. This hinders their effective use and conservation programmes. This study investigates differences in phenological behaviour between bitter and sweet fruited populations and their taxonomic implications. Monthly phenological description data on seven populations of bitter or sweet bush mangos across Benin and Togo were used to assess within and between mango type phenological diversity, to discriminate bitter and sweet trees and to evaluate their responses to environmental factors. The phenological states differentiating bitter and sweet trees were identified and individual trees were classified based on the discriminating phenological characters. Finally, phenological variation was analyzed with time of the year, soil type, type of bush mango tree, and climatic zone. Phenological diversity varies significantly among populations. Bitter and sweet trees have consistently different phenological states. Bitter trees have a lower phenological diversity for all phenological phases throughout the year compared to sweet trees, possibly due to their limited distribution range in the study area. The tree types also differ in their reproductive responses to environmental factors, but did not respond differently to soils. These results support the hypothesis that bitter and sweet trees represent different taxa and we suggest for efficient conservation purpose to consider them as different species.

Estimation of genealogical coancestry in plant species using a pedigree reconstruction algorithm and application to an oil palm breeding population

Explicit pedigree reconstruction by simulated annealing gave reliable estimates of genealogical coancestry in plant species, especially when selfing rate was lower than 0.6, using a realistic number of markers. Genealogical coancestry information is crucial in plant breeding to estimate genetic parameters and breeding values. The approach of Fernández and Toro (Mol Ecol 15:1657-1667, 2006) to estimate genealogical coancestries from molecular data through pedigree reconstruction was limited to species with separate sexes. In this study it was extended to plants, allowing hermaphroditism and monoecy, with possible selfing. Moreover, some improvements were made to take previous knowledge on the population demographic history into account. The new method was validated using simulated and real datasets. Simulations showed that accuracy of estimates was high with 30 microsatellites, with the best results obtained for selfing rates below 0.6. In these conditions, the root mean square error (RMSE) between the true and estimated genealogical coancestry was small (<0.07), although the number of ancestors was overestimated and the selfing rate could be biased. Simulations also showed that linkage disequilibrium between markers and departure from the Hardy-Weinberg equilibrium in the founder population did not affect the efficiency of the method. Real oil palm data confirmed the simulation results, with a high correlation between the true and estimated genealogical coancestry (>0.9) and a low RMSE (<0.08) using 38 markers. The method was applied to the Deli oil palm population for which pedigree data were scarce. The estimated genealogical coancestries were highly correlated (>0.9) with the molecular coancestries using 100 markers. Reconstructed pedigrees were used to estimate effective population sizes. In conclusion, this method gave reliable genealogical coancestry estimates. The strategy was implemented in the software MOLCOANC 3.0.

Estimation of Male Gene Flow from Measures of Nuclear and Female Genetic Differentiation

An approach is provided to estimate male gene flow and the ratio of male to female gene flow, given that there are estimates of diploid, nuclear gene flow and haploid, female gene flow. This approach can be applied to estimates of differentiation (F ST ) from biparentally and maternally inherited markers, assuming the equilibrium island model and equal effective numbers of males and females. Corrections to formulas used previously for California sea lions (González-Suárez M, Flatz R, Aurioles-Gamboa D, Hedrick PW, Gerber LR. 2009. Isolation by distance among California sea lion populations in Mexico: redefining management stocks. Mol Ecol. 18:1088-1099.) and American bison (Halbert ND, Gogan PJP, Hedrick PW, Wahl L, Derr JN. 2012. Genetic population substructure in bison in Yellowstone National Park. J Hered. 103:360-370.) are given and revised values for those species are calculated. The effect of unequal male and female effective population sizes, nonequilibrium conditions, and approximations of differentiation formulas are briefly discussed.

Ecophysiology and genetic structure of polar versus temperate populations of the lichen Cetraria aculeata

We studied polar and temperate samples of the lichen Cetraria aculeata to investigate whether genetical differences between photobionts are correlated with physiological properties of the lichen holobiont. Net photosynthesis and dark respiration (DR) at different temperatures (from 0 to 30 °C) and photon flux densities (from 0 to 1,200 μmol m(-2) s(-1)) were studied for four populations of Cetraria aculeata. Samples were collected from maritime Antarctica, Svalbard, Germany and Spain, representing different climatic situations. Sequencing of the photobiont showed that the investigated samples fall in the polar and temperate clade described in Fernández-Mendoza et al. (Mol Ecol 20:1208-1232, 2011). Lichens with photobionts from these clades differ in their temperature optimum for photosynthesis, maximal net photosynthesis, maximal DR and chlorophyll content. Maximal net photosynthesis was much lower in Antarctica and Svalbard than in Germany and Spain. The difference was smaller when rates were expressed by chlorophyll content. The same is true for the temperature optima of polar (11 °C) and temperate (15 and 17 °C) lichens. Our results indicate that lichen mycobionts may adapt or acclimate to local environmental conditions either by selecting algae from regional pools or by regulating algal cell numbers (chlorophyll content) within the thallus.

Sexing of mid‐incubation avian embryos as a management tool for zoological breeding programs

Skewed sex ratios in zoo breeding programs may require housing single birds of an overrepresented gender, increasing demands on limited resources that could otherwise be diverted to breeding pairs or other important species. The ability to selectively incubate and hatch eggs of a desired sex represents a significant improvement in the long-term management of avian species. This study describes a successful method for in ovo sexing of embryos from stage 30 through 42 of incubation (Hamburger and Hamilton [1951] J Morphol 88:49-92). A 0.01-1 µl blood sample was collected from either the vitelline vessel (VV) or the blood vessels of the chorio-allantoic membrane (CAM) of embryos at stages 14-18 or 30-42, respectively. DNA was isolated from whole blood using the Chelex method (Walsh et al. [1991] Biotechniques 10:506-513; Jensen et al., [2003] Zoo Biol 22:561-571). Sex was determined by PCR amplification using the previously described P2/P8 (Griffiths et al. [1998] Mol Ecol 7:1071-1075) and 1237L/1272H (Kahn et al. [1998] Auk 115:1074-1078) primers or by commercial vendor. Success rate was calculated as the percent of sampled embryos surviving to hatch. Embryos of the undesired sex were not incubated, thus not included in the calculation. There was a considerable difference in success rate when blood was collected from the stage 14-18 VV (0-25%, average 12%) vs. stage 30-42 CAM (33-100%, average 76%). In conclusion, in ovo sexing of embryos between stages 30 and 42 yields acceptable embryo survival rates while providing enough blood for genetic testing.