Scots pine - panmixia and the elusive signal of genetic adaptation.

Scots pine is the foundation species of diverse forested ecosystems across Eurasia and displays remarkable ecological breadth, occurring in environments ranging from temperate rainforests to arid tundra margins. Such expansive distributions can be favored by various demographic and adaptive processes and the interactions between them. To understand the impact of neutral and selective forces on genetic structure in Scots pine, we conducted range-wide population genetic analyses on 2321 trees from 202 populations using genotyping-by-sequencing, reconstructed the recent demography of the species and examined signals of genetic adaptation. We found a high and uniform genetic diversity across the entire range (global FST 0.048), no increased genetic load in expanding populations and minor impact of the last glacial maximum on historical population sizes. Genetic-environmental associations identified only a handful of single-nucleotide polymorphisms significantly linked to environmental gradients. The results suggest that extensive gene flow is predominantly responsible for the observed genetic patterns in Scots pine. The apparent missing signal of genetic adaptation is likely attributed to the intricate genetic architecture controlling adaptation to multi-dimensional environments. The panmixia metapopulation of Scots pine offers a good study system for further exploration into how genetic adaptation and plasticity evolve under gene flow and changing environment.

An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants.

BACKGROUND: The development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture's induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance-Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed. RESULTS: According to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55. CONCLUSIONS: The amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale's GPRE can be increased.

Complete chloroplast genomes of Cerastium alpinum, C. arcticum and C. nigrescens: genome structures, comparative and phylogenetic analysis.

The genus Cerastium includes about 200 species that are mostly found in the temperate climates of the Northern Hemisphere. Here we report the complete chloroplast genomes of Cerastium alpinum, C. arcticum and C. nigrescens. The length of cp genomes ranged from 147,940 to 148,722 bp. Their quadripartite circular structure had the same gene organization and content, containing 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Repeat sequences varied from 16 to 23 per species, with palindromic repeats being the most frequent. The number of identified SSRs ranged from 20 to 23 per species and they were mainly composed of mononucleotide repeats containing A/T units. Based on Ka/Ks ratio values, most genes were subjected to purifying selection. The newly sequenced chloroplast genomes were characterized by a high frequency of RNA editing, including both C to U and U to C conversion. The phylogenetic relationships within the genus Cerastium and family Caryophyllaceae were reconstructed based on the sequences of 71 protein-coding genes. The topology of the phylogenetic tree was consistent with the systematic position of the studied species. All representatives of the genus Cerastium were gathered in a single clade with C. glomeratum sharing the least similarity with the others.

Molecular structure, comparative and phylogenetic analysis of the complete chloroplast genome sequences of weedy rye Secale cereale ssp. segetale.

The complete chloroplast genome of Secale cereale ssp. segetale (Zhuk.) Roshev. (Poaceae: Triticeae) was sequenced and analyzed to better use its genetic resources to enrich rye and wheat breeding. The study was carried out using the following methods: DNA extraction, sequencing, assembly and annotation, comparison with other complete chloroplast genomes of the five Secale species, and multigene phylogeny. As a result of the study, it was determined that the chloroplast genome is 137,042 base pair (bp) long and contains 137 genes, including 113 unique genes and 24 genes which are duplicated in the IRs. Moreover, a total of 29 SSRs were detected in the Secale cereale ssp. segetale chloroplast genome. The phylogenetic analysis showed that Secale cereale ssp. segetale appeared to share the highest degree of similarity with S. cereale and S. strictum. Intraspecific diversity has been observed between the published chloroplast genome sequences of S. cereale ssp. segetale. The genome can be accessed on GenBank with the accession number (OL688773).

The comparison of polymorphism among Avena species revealed by retrotransposon-based DNA markers and soluble carbohydrates in seeds.

Here, we compared the polymorphism among 13 Avena species revealed by the iPBS markers and soluble carbohydrate profiles in seeds. The application of seven iPBS markers generated 83 bands, out of which 20.5% were polymorphic. No species-specific bands were scored. Shannon's information index (I) and expected heterozygosity (He) revealed low genetic diversity, with the highest values observed for A. nuda (I = 0.099; He = 0.068). UPGMA clustering of studied Avena accessions and PCoA results showed that the polyploidy level is the main grouping criterion. High-resolution gas chromatography revealed that the studied Avena accessions share the same composition of soluble carbohydrates, but significant differences in the content of total (5.30-22.38 mg g-1 of dry weight) and particular sugars among studied samples were observed. Sucrose appeared as the most abundant sugar (mean 61.52% of total soluble carbohydrates), followed by raffinose family oligosaccharides (31.23%), myo-inositol and its galactosides (6.16%), and monosaccharides (1.09%). The pattern of interspecific variation in soluble carbohydrates, showed by PCA, was convergent to that revealed by iPBS markers. Thus, both methods appeared as a source of valuable data useful in the characterization of Avena resources or in the discussion on the evolution of this genus.

Genetic variation of Cerastium alpinum L. from Babia Góra, a critically endangered species in Poland.

Babia Góra massif is the only site of occurrence of the Cerastium alpinum L. in Poland, an arctic-alpine perennial plant with a wide distribution in North America, northwestern Asia, and Europe. To determine whether the isolated Polish populations are genetically distinct, we have performed an evaluation of C. alpinum from Babia Góra with the use of iPBS markers. A total number of 133 individuals of C. alpinum from seven populations representing four localizations of the species were analyzed, i.e., from Babia Góra (Poland), Alps (Switzerland), Nuolja massif (Sweden), and Kaffiøyra (Svalbard, Norway). Genetic analysis of all C. alpinum samples using eight PBS primers identified 262 bands, 79.4% of which were polymorphic. iPBS markers revealed low genetic diversity (average He = 0.085) and high population differentiation (FST = 0.617). AMOVA results confirmed that the majority of the genetic variation (62%) was recorded among populations. The grouping revealed by PCoA showed that C. alpinum from Svalbard is the most diverged population, C. alpinum from Switzerland and Sweden form a pair of similar populations, whereas C. alpinum from the Babia Góra form a heterogeneous group of four populations. Results of isolation by distance analysis suggested that the spatial distance is the most probable cause of the observed differentiation among populations. Although significant traces of a bottleneck effect were noted for all populations of C. alpinum from Babia Góra, the populations still maintain a low but significant level of genetic polymorphism. These results are of great importance for developing conservation strategies for this species in Poland.

S-Adenosyl-L-Methionine and Cu(II) Impact Green Plant Regeneration Efficiency.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Molecular Diversity and Phylogeny Reconstruction of Genus Colobanthus (Caryophyllaceae) Based on Mitochondrial Gene Sequences.

Mitochondrial genomes have become an interesting object of evolutionary and systematic study both for animals and plants, including angiosperms. Although the framework of the angiosperm phylogeny was built on the information derived from chloroplast and nuclear genes, mitochondrial sequences also revealed their usefulness in solving the phylogenetic issues at different levels of plant systematics. Here, we report for the first time the complete sequences of 26 protein-coding genes of eight Colobanthus species (Caryophyllaceae). Of these, 23 of them represented core mitochondrial genes, which are directly associated with the primary function of that organelle, and the remaining three genes represented a facultative set of mitochondrial genes. Comparative analysis of the identified genes revealed a generally high degree of sequence conservation. The Ka/Ks ratio was <1 for most of the genes, which indicated purifying selection. Only for rps12 was Ka/Ks > 1 in all studied species, suggesting positive selection. We identified 146−165 potential RNA editing sites in genes of the studied species, which is lower than in most angiosperms. The reconstructed phylogeny based on mitochondrial genes was consistent with the taxonomic position of the studied species, showing the separate character of the family Caryophyllaceae and close relationships between all studied Colobanthus species, with C. lycopodioides sharing less similarity.

Characterization and phylogenetic analysis of the complete mitochondrial genome of the pathogenic fungus Ilyonectria destructans.

Ilyonectria destructans is a pathogenic fungus causing root rot and other symptoms on trees and many crops. This paper analyses the mitochondrial genome of I. destructans and compares it with other published Nectriaceae mitogenomes. The I. destructans mitogenome appears as a circular DNA molecule of 42,895 bp and an overall GC content of 28.23%. It contains 28 protein-coding genes (15 core protein genes and 13 free-standing ORFs), two rRNAs and 27 tRNAs. The gene content and order were found to be conserved in the mitogenome of I. destructans and other Nectriaceae, although the genome size varies because of the variation in the number and length of intergenic regions and introns. For most core protein-coding genes in Nectriaceae species, Ka/Ks < 1 indicates purifying selection. Among some Nectriaceae representatives, only the rps3 gene was found under positive selection. Phylogenetic analyses based on nucleotide sequences of 15 protein-coding genes divided 45 Hypocreales species into six major clades matching the families Bionectriaceae, Cordycipitaceae, Clavicipitaceae, Ophiocordycipitaceae, Hypocreaceae and Nectriaceae. I. destructans appeared as a sister species to unidentified Ilyonectia sp., closely related to C. ilicicola, N. cinnabarina and a clad of ten Fusarium species and G. moniliformis. The complete mitogenome of I. destructans reported in the current paper will facilitate the study of epidemiology, biology, genetic diversity of the species and the evolution of family Nectriace and the Hypocreales order.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture.

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

Retrotransposon-based genetic diversity of Deschampsia antarctica Desv. from King George Island (Maritime Antarctic).

Deschampsia antarctica Desv. can be found in diverse Antarctic habitats which may vary considerably in terms of environmental conditions and soil properties. As a result, the species is characterized by wide ecotypic variation in terms of both morphological and anatomical traits. The species is a unique example of an organism that can successfully colonize inhospitable regions due to its phenomenal ability to adapt to both the local mosaic of microhabitats and to general climatic fluctuations. For this reason, D. antarctica has been widely investigated in studies analyzing morphophysiological and biochemical responses to various abiotic stresses (frost, drought, salinity, increased UV radiation). However, there is little evidence to indicate whether the observed polymorphism is accompanied by the corresponding genetic variation. In the present study, retrotransposon-based iPBS markers were used to trace the genetic variation of D. antarctica collected in nine sites of the Arctowski oasis on King George Island (Western Antarctic). The genotyping of 165 individuals from nine populations with seven iPBS primers revealed 125 amplification products, 15 of which (12%) were polymorphic, with an average of 5.6% polymorphic fragments per population. Only one of the polymorphic fragments, observed in population 6, was represented as a private band. The analyzed specimens were characterized by low genetic diversity (uHe = 0.021, I = 0.030) and high population differentiation (F ST = 0.4874). An analysis of Fu's F S statistics and mismatch distribution in most populations (excluding population 2, 6 and 9) revealed demographic/spatial expansion, whereas significant traces of reduction in effective population size were found in three populations (1, 3 and 5). The iPBS markers revealed genetic polymorphism of D. antarctica, which could be attributed to the mobilization of random transposable elements, unique features of reproductive biology, and/or geographic location of the examined populations.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium.

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Evolutionary dynamics of the chloroplast genome sequences of six Colobanthus species.

The complete plastome sequences of six species were sequenced to better understand the evolutionary relationships and mutation patterns in the chloroplast genome of the genus Colobanthus. The length of the chloroplast genome sequences of C. acicularis, C. affinis, C. lycopodioides, C. nivicola, C. pulvinatus and C. subulatus ranged from 151,050 to 151,462 bp. The quadripartite circular structure of these genome sequences has the same overall organization and gene content with 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames. A total of 153 repeat sequences were revealed. Forward repeats were dominant, whereas complementary repeats were found only in C. pulvinatus. The mononucleotide SSRs composed of A/T units were most common, and hexanucleotide SSRs were detected least often. Eleven highly variable regions which could be utilized as potential markers for phylogeny reconstruction, species identification or phylogeography were identified within Colobanthus chloroplast genomes. Seventy-three protein-coding genes were used in phylogenetic analyses. Reconstructed phylogeny was consistent with the systematic position of the studied species, and the representatives of the same genus were grouped in one clade. All studied Colobanthus species formed a single group and C. lycopodioides was least similar to the remaining species.

rps3 as a Candidate Mitochondrial Gene for the Molecular Identification of Species from the Colletotrichum acutatum Species Complex.

Colletotrichum species form one of the most economically significant groups of pathogenic fungi and lead to significant losses in the production of major crops-in particular, fruits, vegetables, ornamental plants, shrubs, and trees. Members of the genus Colletotrichum cause anthracnose disease in many plants. Due to their considerable variation, these fungi have been widely investigated in genetic studies as model organisms. Here, we report the complete mitochondrial genome sequences of four Colletotrichum species (C. fioriniae, C. lupini, C. salicis, and C. tamarilloi). The reported circular mitogenomes range from 30,020 (C. fioriniae) to 36,554 bp (C. lupini) in size and have identical sets of genes, including 15 protein-coding genes, two ribosomal RNA genes, and 29 tRNA genes. All four mitogenomes are characterized by a rather poor repetitive sequence content with only forward repeat representatives and a low number of microsatellites. The topology of the phylogenetic tree reflects the systematic positions of the studied species, with representatives of each Colletotrichum species complex gathered in one clade. A comparative analysis reveals consistency in the gene composition and order of Colletotrichum mitogenomes, although some highly divergent regions are also identified, like the rps3 gene which appears as a source of potential diagnostic markers for all studied Colletotrichum species.

Retrotransposon-based genetic variation of Poa annua populations from contrasting climate conditions.

BACKGROUND: Poa annua L. is an example of a plant characterized by abundant, worldwide distribution from polar to equatorial regions. Due to its high plasticity and extraordinary expansiveness, P. annua is considered an invasive species capable of occupying and surviving in a wide range of habitats including pioneer zones, areas intensively transformed by human activities, remote subarctic meadows and even the Antarctic Peninsula region. METHODS: In the present study, we evaluated the utility of inter-primer binding site (iPBS) markers for assessing the genetic variation of P. annua populations representing contrasting environments from the worldwide range of this species. The electrophoretic patterns of polymerase chain reaction products obtained for each individual were used to estimate the genetic diversity and differentiation between populations. RESULTS: iPBS genotyping revealed a pattern of genetic variation differentiating the six studied P. annua populations characterized by their different climatic conditions. According to the analysis of molecular variance, the greatest genetic variation was recorded among populations, whereas 41.75% was observed between individuals within populations. The results of principal coordinates analysis (PCoA) and model-based clustering analysis showed a clear subdivision of analyzed populations. According to PCoA, populations from Siberia and the Kola Peninsula were the most different from each other and showed the lowest genetic variability. The application of STRUCTURE software confirmed the unique character of the population from the Kola Peninsula. DISCUSSION: The lowest variability of the Siberia population suggested that it was subjected to genetic drift. However, although demographic expansion was indicated by negative values of Fu's FS statistic and analysis of mismatch distribution, it was not followed by significant traces of a bottleneck or a founder effect. For the Antarctic population, the observed level of genetic variation was surprisingly high, despite the observed significant traces of bottleneck/founder effect following demographic expansion, and was similar to that observed in populations from Poland and the Balkans. For the Antarctic population, the multiple introduction events from different sources are considered to be responsible for such an observation. Moreover, the results of STRUCTURE and PCoA showed that the P. annua from Antarctica has the highest genetic similarity to populations from Europe. CONCLUSIONS: The observed polymorphism should be considered as a consequence of the joint influence of external abiotic stress and the selection process. Environmental changes, due to their ability to induce transposon activation, lead to the acceleration of evolutionary processes through the production of genetic variability.

The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species.

Colobanthus apetalus is a member of the genus Colobanthus, one of the 86 genera of the large family Caryophyllaceae which groups annual and perennial herbs (rarely shrubs) that are widely distributed around the globe, mainly in the Holarctic. The genus Colobanthus consists of 25 species, including Colobanthus quitensis, an extremophile plant native to the maritime Antarctic. Complete chloroplast (cp) genomes are useful for phylogenetic studies and species identification. In this study, next-generation sequencing (NGS) was used to identify the cp genome of C. apetalus. The complete cp genome of C. apetalus has the length of 151,228 bp, 36.65% GC content, and a quadripartite structure with a large single copy (LSC) of 83,380 bp and a small single copy (SSC) of 17,206 bp separated by inverted repeats (IRs) of 25,321 bp. The cp genome contains 131 genes, including 112 unique genes and 19 genes which are duplicated in the IRs. The group of 112 unique genes features 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs). A total of 12 forward repeats, 10 palindromic repeats, five reverse repeats and three complementary repeats were detected. In addition, a simple sequence repeat (SSR) analysis revealed 41 (mono-, di-, tri-, tetra-, penta- and hexanucleotide) SSRs, most of which were AT-rich. A detailed comparison of C. apetalus and C. quitensis cp genomes revealed identical gene content and order. A phylogenetic tree was built based on the sequences of 76 protein-coding genes that are shared by the eleven sequenced representatives of Caryophyllaceae and C. apetalus, and it revealed that C. apetalus and C. quitensis form a clade that is closely related to Silene species and Agrostemma githago. Moreover, the genus Silene appeared as a polymorphic taxon. The results of this study expand our knowledge about the evolution and molecular biology of Caryophyllaceae.

How much of the invader's genetic variability can slip between our fingers? A case study of secondary dispersal of Poa annua on King George Island (Antarctica).

We studied an invasion of Poa annua on King George Island (Maritime Antarctic). The remoteness of this location, its geographic isolation, and its limited human traffic provided an opportunity to trace the history of an invasion of the species. Poa annua was recorded for the first time at H. Arctowski Polish Antarctic Station in the austral summer of 1985/6. In 2008/9, the species was observed in a new locality at the Ecology Glacier Forefield (1.5 km from "Arctowski"). We used AFLP to analyze the genetic differences among three populations of P. annua: the two mentioned above (Station and Forefield) and the putative origin of the introduction, Warsaw (Poland). There was 38% genetic variance among the populations. Pairwise ФPT was 0.498 between the Forefield and Warsaw populations and 0.283 between Warsaw and Station. There were 15 unique bands in the Warsaw population (frequency from 6% to 100%) and one in the Station/Forefield populations (which appears in all analyzed individuals from both populations). The Δ(K) parameter indicated two groups of samples: Warsaw/Station and Forefield. As indicated by Fu's Fs statistics and an analysis of mismatch distribution, the Forefield population underwent a bottleneck and/or founder effect. The Forefield population was likely introduced by secondary dispersal from the Station population.