Frugivorous birds mediate sex‐biased facilitation in a dioecious nurse plant

Abstract. Facilitation by dispersal occurs if the nurse plant acts as a focus which is actively selected by seed dispersers and enhances the fitness of the facilitated plant. Sex‐biased facilitation may be produced if seed dispersers tend to concentrate the seeds under female, fruit‐bearing plants of dioecious species more often than under conspecific males. Juniperus sabina is a dioecious shrub with a prostrate growth form from Mediterranean high mountains that modifies many microhabitat characteristics related to seedling establishment and survival. Soil water availability, maximum soil temperature in summer, organic matter and total nitrogen content, were different on open ground as compared with beneath J. sabina shrubs, irrespective of its sex. Other studied characteristics such as soil bulk density and soil compaction after rain did not differ between the microhabitats considered. Some species, such as Juniperus communis, Pinus nigra, Helleborus foetidus and Euphorbia nicaeensis, are spatially associated to J. sabina shrubs, strongly suggesting a facilitative role. The anemochorous P. nigra and myrme‐chorous H. foetidus and E. nicaeensis did not associate preferentially to any sex of J. sabina. Only J. communis, an endozoochorous species sharing the same bird dispersers as J. sabina, presented a female‐biased spatial association with the nurse plant. Seed dispersal mediated by birds attracted by the fruit‐rewarding females of J. sabina explains the sex‐biased spatial pattern of Juniperus communis.     

Hybridization between two parapatric ranid frog species in the northern Sierra Nevada,California, USA

Contact zones between species provide a unique opportunity to test whether taxa can hybridize or not. Cross‐breeding or hybridization between closely related taxa can promote gene flow (introgression) between species, adaptation, or even speciation. Though hybridization events may be short‐lived and difficult to detect in the field, genetic data can provide information about the level of introgression between closely related taxa. Hybridization can promote introgression between species, which may be an important evolutionary mechanism for either homogenization (reversing initial divergence between species) or reproductive isolation (potentially leading to speciation). Here, we used thousands of genetic markers from nuclear DNA to detect hybridization between two parapatric frog species (Rana boylii and Rana sierrae) in the Sierra Nevada of California. Based on principal components analysis, admixture, and analysis of heterozygosity at species diagnostic SNPs, we detected two F1 hybrid individuals in the Feather River basin, as well as a weak signal of introgression and gene flow between the frog species compared with frog populations from two other adjacent watersheds. This study provides the first documentation of hybridization and introgression between these two species, which are of conservation concern.     

Life history of Juniperus sabina L. adapted to the sand shifting environment in the Mu Us Sandy Land,China: A review

We have reviewed publications on the physiological and ecological features of the growth and regeneration processes of Juniperus sabina L. which grows in semiarid sandy land in the Ordos plateau in northern China where desertification has progressed over time. J. sabina is a key native plant species used for ecological restoration in this region. The life history of J. sabina in this sandy land that has been revealed through this review includes several unique features: (1) both vegetative and seed propagations are observed, but seed propagation is not successful in the location where the mature J. sabina stands. Instead, seed propagation can occur at a different place with different landscapes from the mature stands. (2) Nurse plants play an important role in providing the microclimatic environment necessary for the growth of J. sabina seedlings and young plants. (3) During the horizontal and vertical growth processes of the J. sabina patch, the root system was affected by burial in shifting sand and consequently acquired greater access to the water supply in deeper soil horizons, which could support larger growth. These characteristics suggested that the regeneration by seed propagation and growth strategy of J. sabina in this region was strongly affected by sand movement and the landscape that is generated by sand movement.

     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Geographic mosaic of symbiont selectivity in a genus of epiphytic cyanolichens

In symbiotic systems, patterns of symbiont diversity and selectivity are crucial for the understanding of fundamental ecological processes such as dispersal and establishment. The lichen genus Nephroma (Peltigerales, Ascomycota) has a nearly cosmopolitan distribution and is thus an attractive model for the study of symbiotic interactions over a wide range of spatial scales. In this study, we analyze the genetic diversity of Nephroma mycobionts and their associated Nostoc photobionts within a global framework. The study is based on Internal Transcribed Spacer (ITS) sequences of fungal symbionts and tRNA^Leu (UAA) intron sequences of cyanobacterial symbionts. The full data set includes 271 Nephroma and 358 Nostoc sequences, with over 150 sequence pairs known to originate from the same lichen thalli. Our results show that all bipartite Nephroma species associate with one group of Nostoc different from Nostoc typically found in tripartite Nephroma species. This conserved association appears to have been inherited from the common ancestor of all extant species. While specific associations between some symbiont genotypes can be observed over vast distances, both symbionts tend to show genetic differentiation over wide geographic scales. Most bipartite Nephroma species share their Nostoc symbionts with one or more other fungal taxa, and no fungal species associates solely with a single Nostoc genotype, supporting the concept of functional lichen guilds. Symbiont selectivity patterns within these lichens are best described as a geographic mosaic, with higher selectivity locally than globally. This may reflect specific habitat preferences of particular symbiont combinations, but also the influence of founder effects.     

Habitat predictors of genetic diversity for two sympatric wetland‐breeding amphibian species

Population genetic diversity is widely accepted as important to the conservation and management of wildlife. However, habitat features may differentially affect evolutionary processes that facilitate population genetic diversity among sympatric species. We measured genetic diversity for two pond‐breeding amphibian species (Dwarf salamanders, Eurycea quadridigitata; and Southern Leopard frogs, Lithobates sphenocephalus) to understand how habitat characteristics and spatial scale affect genetic diversity across a landscape. Samples were collected from wetlands on a longleaf pine reserve in Georgia. We genotyped microsatellite loci for both species to assess population structures and determine which habitat features were most closely associated with observed heterozygosity and rarefied allelic richness. Both species exhibited significant population genetic structure; however, structure in Southern Leopard frogs was driven primarily by one outlier site. Dwarf salamander allelic richness was greater at sites with less surrounding road area within 0.5 km and more wetland area within 1.0 and 2.5 km, and heterozygosity was greater at sites with more wetland area within 0.5 km. In contrast, neither measure of Southern Leopard frog genetic diversity was associated with any habitat features at any scale we evaluated. Genetic diversity in the Dwarf salamander was strongly associated with land cover variables up to 2.5 km away from breeding wetlands, and/or results suggest that minimizing roads in wetland buffers may be beneficial to the maintenance of population genetic diversity. This study suggests that patterns of genetic differentiation and genetic diversity have associations with different habitat features across different spatial scales for two syntopic pond‐breeding amphibian species.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Hidden diversity and cryptic speciation refute cosmopolitan distribution in Caprella penantis (Crustacea: Amphipoda: Caprellidae)

Caprella penantis is considered a cosmopolitan species and one of the most challenging caprellids in taxonomic terms because of its remarkable intraspecific morphological variation. This study examined DNA sequences from mitochondrial (COI) and nuclear (18S) markers together with morphological data from 25 localities of C. penantis, and closely related species Caprella dilatata and Caprella andreae, all traditionally considered part of the old ‘acutifrons’ complex. The large genetic divergence and reciprocally allopatric distributions point to the existence of a species complex of at least four species, of which one is reported as a cryptic species. This study provides the first evidence of cryptic speciation in the family Caprellidae, and questions the validity of some traditional morphological characters used to delimit species in the genus Caprella. Our results are consistent with the idea that main factors were probably isolation by distance and ecological traits, promoting diversification in C. penantis. The strong genetic structure reported for this species in the Iberian Peninsula and Moroccan coasts also suggests restriction to dispersal as well as the presence of refugial areas. These results highlight the utility of the COI and 18S genes in combination with morphological characters for shedding light on systematic questions in caprellids, and patterns of genetic connectivity.     

Genetic variation in Tertiary relics: The case of eastern‐Mediterranean Abies (Pinaceae)

The eastern‐Mediterranean Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern‐Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the Structure analysis and distance‐based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were distinct but less differentiated. ABC simulations were run for a set of scenarios of phylogeny and past demographic changes. For A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization, the same is true for Abies equi‐trojani: both they represent evolutionary branches of Abies bornmuelleriana.     

Development and characterization of EST‐SSR markers in an East Asian temperate plant genus Diabelia (Caprifoliaceae)

A set of expressed sequence tag (EST) simple sequence repeat (SSR) markers were developed and characterized using next‐generation sequencing technology for the genus Diabelia (Caprifoliaceae). De novo assembly of RNA‐seq reads resulted in 58 669 contigs with the N50 length of 1211 bp. A total of 2746 contigs were identified to harbor SSR motifs, of which 48 primer pairs were designed and 11 were shown to be polymorphic across three morphospecies of Diabelia. When evaluated with 30 individuals, the number of alleles per locus ranged from 2 to 11 and the expected heterozygosity varied from 0.399 to 0.873, respectively. Distance‐based clustering indicated that the EST‐SSR markers can provide sufficient power to distinguish the three species (or populations). These markers will be useful for evaluating the range‐wide genetic diversity of each species and examining genetic divergence and gene flow between the three species.     

Development and characterization of 20 polymorphic microsatellite markers for Livistona chinensis R. Br. var. subglobosa Becc., a fan palm in the Ryukyu Archipelago,Japan

Livistona chinensis R. Br. var. subglobosa Becc. is a subtropical fan palm distributed mainly in the islands of the Ryukyu Archipelago, Japan. Some small populations, separated by a few 100 km from the main species range, are also established on islands around northern Kyushu and Cape Ashizuri, Shikoku. Most of those are designated as national or prefectural natural treasures. We developed 20 microsatellite markers for this species and characterized them for polymorphisms within six populations. The number of alleles per locus ranged from two to nine, with an average of 4.2; the expected heterozygosity ranged from 0.16 to 0.61, with an average of 0.46. These markers will be useful for investigating disjunct range formation within L. chinensis var. subglobosa and the origins of the remote populations, providing crucial information for conservation of the populations.     

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae)

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A–G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus‐wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on ‘best match’ analysis, the combination of matK+ITS2 was best, while based on ‘all species barcodes’ analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult.     

Testing the effects of heterozygosity on growth rate plasticity in the seaweed Gracilaria chilensis (Rhodophyta)

Heterozygosity has been positively associated with fitness and population survival. However, the relationship between heterozygosity and adaptive phenotypic plasticity (i.e., plasticity which results in fitness homeostasis or improvement in changing environments) is unclear and has been poorly explored in seaweeds. In this study, we explored this relationship in the clonal red seaweed, Gracilaria chilensis by conducting three growth rate plasticity experiments under contrasting salinity conditions and by measuring heterozygosity with five microsatellite DNA markers. Firstly, we compared growth rate plasticity between the haploid and diploid phases. Secondly, we compared growth rate plasticity between diploids with different numbers of heterozygous loci. Finally, we compared growth rate plasticity between diploid plants from two populations that are expected to exhibit significant differences in heterozygosity. We found that, (i) diploids displayed a higher growth rate and lower growth rate plasticity than haploids, (ii) diploids with a higher number of heterozygous loci displayed lower growth rate plasticity than those exhibiting less heterozygosity, and (iii) diploid sporophytes from the population with higher heterozygosity displayed lower growth rate plasticity than those with lower heterozygosity. Accordingly, this study suggests that heterozygosity is inversely related to growth rate plasticity in G. chilensis. However, better genetic tools in seaweeds are required for a more definitive conclusion on the relationship between heterozygosity and phenotypic plasticity.     

No correlation between multi‐locus heterozygosity and fitness in the common buzzard despite heterozygote advantage for plumage colour

Correlations between heterozygosity and fitness are frequently found but rarely well understood. Fitness can be affected by single loci of large effect which correlate with neutral markers via linkage disequilibrium, or as a result of variation in genome‐wide heterozygosity following inbreeding. We explored these alternatives in the common buzzard, a raptor species in which three colour morphs differ in their lifetime reproductive success. Using 18 polymorphic microsatellite loci, we evaluated potential genetic differences among the morphs which may lead to subpopulation structuring and tested for correlations between three fitness‐related traits and heterozygosity, both genome wide and at each locus separately. Despite their assortative mating pattern, the buzzard morphs were found to be genetically undifferentiated. Multilocus heterozygosity was only found to be correlated with a single fitness‐related trait, infection with the blood parasite, Leucocytozoon buteonis, and this was via interactions with vole abundance and age. One locus also showed a significant relationship with blood parasite infection and ectoparasite infestation. The vicinity of this locus contains two genes, one of which is potentially implicated in the immune system of birds. We conclude that genome‐wide heterozygosity is unlikely to be a major determinant of parasite burden and body condition in the polymorphic common buzzard.     

Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.     

Species identification and comparative population genetics of four coastal houndsharks based on novel NGS‐mined microsatellites

The common smooth‐hound (Mustelus mustelus ) is the topmost bio‐economically and recreationally important shark species in southern Africa, western Africa, and Mediterranean Sea. Here, we used the Illumina HiSeq? 2000 next‐generation sequencing (NGS ) technology to develop novel microsatellite markers for Mustelus mustelus . Two microsatellite multiplex panels were constructed from 11 polymorphic loci and characterized in two populations of Mustelus mustelus representative of its South African distribution. The markers were then tested for cross‐species utility in Galeorhinus galeus , Mustelus palumbes , and Triakis megalopterus , three other demersal coastal sharks also subjected to recreational and/or commercial fishery pressures in South Africa. We assessed genetic diversity (N _A, A _R, H _O, H _E, and PIC) and differentiation (F _ST and D _est) for each species and also examined the potential use of these markers in species assignment. In each of the four species, all 11 microsatellites were variable with up to a mean N _A of 8, A _R up to 7.5, H _E and PIC as high as 0.842. We were able to reject genetic homogeneity for all species investigated here except for T . megalopterus . We found that the panel of the microsatellite markers developed in this study could discriminate between the study species, particularly for those that are morphologically very similar. Our study provides molecular tools to address ecological and evolutionary questions vital to the conservation and management of these locally and globally exploited shark species.     

Peripheral genetic structure of Helicoverpa zea indicates asymmetrical panmixia

Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F‐statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low F_ST (0.01 to 0.02) and high F_IS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.     

Isolation and characterization of microsatellite loci in the threatened brook lamprey Lethenteron sp. N

A microsatellite‐enriched genomic library was obtained for the threatened brook lamprey, Lethenteron sp. N, and from it 16 dinucleotide markers were successfully isolated and characterized. These markers were found to have between 1 and 6 alleles with heterozygosity ranging from zero to 0.79. Cross‐species amplification was successful, with eight loci amplifying products in the counterpart cryptic species, Lethenteron sp. S, as well as 15 loci in Lethenteron japonicum. One out of 10 primer pairs previously reported also amplified products in all the three species.     

Eight new microsatellite loci for the critically endangered fire‐bellied toad Bombina bombina and their cross‐species applicability among anurans

We describe eight new microsatellite loci for the critically endangered fire‐bellied toad, Bombina bombina. Seven of them are polymorphic with two to seven alleles per locus, an expected heterozygosity between 0.41 and 0.8, and an observed heterozygosity between 0.27 and 0.7. The yield of new loci was relatively low, presumably due to mildly repetitive sequence motifs in microsatellite flanking regions. As typical for anurans, cross‐species amplification was limited (here, to congeners Bombina orientalis and Bombina variegata). Combining these new loci with those already available provides a reasonable number of loci for population studies and pedigree analysis in Bombina.     

Is DNA barcoding child's play? Science education and the utility of DNA barcoding for the discrimination of UK tree species

We present the findings of a DNA barcoding study of the UK tree flora, implemented as part of an innovative, research‐based science education programme called ‘Tree School’. The UK tree flora comprises native and introduced species, and is a taxonomically diverse study group for the exploration of the potential and limitations of DNA barcoding. The children participating in the project collected voucher specimens and generated DNA barcode sequences from trees and shrubs found in the grounds and surrounding woodlands of a residential field centre in Dorset, UK. We assessed the potential of rbcL and matK markers for amplification and DNA sequencing success and for species discrimination among the 67 tree and shrub species included in this study. Although we achieved 100% PCR amplification and sequencing success for rbcL and matK, mononucleotide repeats affected sequence quality in matK for some taxonomic groups (e.g. Rosaceae). Species discrimination success ranged from 65% to 71% using tree‐based methods to 86% using BLASTN. The occurrence of known hybrids (diploid and polyploid) and their progenitors on the study site reduced the overall species discrimination success for both loci. This study demonstrates that, even in a floristic context, rbcL and matK alone are insufficient for the discrimination of UK tree species, especially where taxonomically complex groups are present. From a science education perspective, DNA barcoding represents a compelling and accessible platform for the engagement of non‐experts in ongoing research, providing an opportunity for them to contribute authentic scientific data to an international research campaign.