Geographic patterns of genetic variation in nuclear and chloroplast genomes of two related oaks (<Emphasis Type="Italic">Quercus aliena</Emphasis> and <Emphasis Type="Italic">Q. serrata</Emphasis>) in Japan: implications for seed and seedling transfer

In this study, we assessed geographic patterns of genetic variations in nuclear and chloroplast genomes of two related native oaks in Japan, Quercus aliena and Q. serrata, in order to facilitate development of genetic guidelines for transfer of planting stocks for each species. A total of 12 populations of Q. aliena and 44 populations of Q. serrata were analyzed in this study. Genotyping of nuclear microsatellites in Q. aliena was done with only nine populations (n = 212) due to limited numbers of individuals in two populations, while all 12 populations (n = 89) were used in sequencing chloroplast DNA (cpDNA). In Q. serrata, 43 populations (n = 1032) were genotyped by nuclear microsatellite markers, while cpDNA of 44 populations (n = 350) was sequenced. As anticipated, geographic patterns detected in the variations of Q. aliena’s nuclear genome and its chloroplast haplotype distribution clearly distinguished northern and southern groups of populations. However, those of Q. serrata were inconsistent. The geographic distribution of its chloroplast haplotypes tends to show the predicted differentiation between northern and southern lineages, but geographic signals in the genetic structure of its nuclear microsatellites are weak. Therefore, treating northern and southern regions of Japan as genetically distinct transferrable zones for planting stocks is highly warranted for Q. aliena. For Q. serrata, the strong NE-SW geographic structure of cpDNA should be considered.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.     

Development and Characterization of Microsatellite Markers for Genetic Analysis of the Swimming Crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

This study isolated and characterized 11 novel microsatellite markers for the commercially important swimming crab species, Portunus trituberculatus. Genetic diversity and population structure of two populations of P. trituberculatus in the East China Sea were assessed using these loci. The microsatellite markers produced 242 alleles, varying from 17 to 26 alleles per locus. In all the samples, the range of heterozygosity was 0.6324–0.9403 (observed) and 0.8998–0.9547 (expected). An F-statistic analysis revealed low genetic differentiation between the populations (mean F _ST = 0.0197), with 98% of the variation resulting from the within-population component. In addition, cross-amplification was tested in two other portunid species, and we found that many loci yielded useful information. The high degree of polymorphism exhibited by the 11 microsatellites suggests that these markers will be useful for both aquaculture and studies of natural populations of the genus.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.     

Population genetic analyses of the endangered alpine <Emphasis Type="Italic">Sinadoxa corydalifolia</Emphasis> (Adoxaceae) provide insights into future conservation

With increasing temperature and anthropogenic activity, endangered alpine species in the high altitudes of the Qinghai-Tibet Plateau face high risk of extinction; however, they have received little attention in the past. In this study, we used 12 nuclear and nine chloroplast microsatellites (simple sequence repeats, SSR) to assess genetic diversity within and among the only two populations of the highly endangered alpine species Sinadoxa corydalifolia (Adoxaceae). We identified only one individual exhibiting clonal reproduction across all 160 extant plants. The levels of genetic variability were estimated to be very low, with the allele number N_a = 3.2 and the expected heterozygosity H_e = 0.368. The genetic differentiation is extremely high between the two regional populations (F_ST = 0.214), with a limited rate of gene flow in the recent past. In addition, numerous endemic alleles were found for each subpopulation within each population. Our analyses suggest that it is critical not only to conserve all surviving individuals of the two populations in situ but also to mediate gene flow artificially between subpopulations within each population in this endangered species.     

Genetic consequences of anthropogenic disturbances and population fragmentation in <Emphasis Type="Italic">Acacia senegal</Emphasis>

Acacia senegal is endemic to dry forest and woodland ecosystems of Sub-Saharan Africa and provides both ecological and socio-economic benefits. However, these ecosystems are threatened by escalating human disturbances and fragmentation. To investigate the human impacts on genetic diversity and structure of A. senegal, we studied genetic variability and differentiation of 330 individual trees from 11 natural A. senegal populations, grouped into lightly and heavily disturbed, using 12 polymorphic nuclear microsatellite markers. Gene diversity (H _E ) ranged from H _E = 0.570 to H _E = 0.632. Significant differences (P < 0.05) between the levels of disturbances are reported for mean gene diversity, number of alleles and allelic richness with lightly disturbed populations showing higher values. Overall, the indirect estimates of average outcrossing rates ranged from 0.794 (Kiserian) to 0.999 (Kampi ya Moto) with a mean of 0.997 suggesting a predominantly outcrossing species. There was no significant relationship (P > 0.05) detected between genetic and geographic distances, showing lack of isolation by distance. Analysis of population structure using unweighted pair group method with arithmetic mean and Bayesian model suggests presence of three gene pools as most probable, although most individuals showed mixed ancestry. The diversity and genetic structure reported in this study revealed negative impacts of human disturbance on A. senegal within this ecosystem. We recommend in-situ conservation strategies to safeguard the woodland ecosystem from further deforestation.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Genetic Differentiation of Colombian Populations of <Emphasis Type="Italic">Anopheles darlingi</Emphasis> 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.     

Potential of cross-species microsatellite markers to assess population genetics of the endemic,endangered Nilgiri tahr (<Emphasis Type="Italic">Nilgiritragus hylocrius</Emphasis>)

Nilgiri tahr, the only wild representative of the Caprinae subfamily in Southern India, is endangered due to population decline, decreasing range size and limited geographical distribution, which together with habitat loss and fragmentation, further reduce its long-term viability. Planning conservation and management strategies to rehabilitate the species will require information on its population status and genetics. With an objective to assess the population genetics of Nilgiri tahr, we identified a panel of polymorphic microsatellite markers that amplify across the Caprinae species. We screened 50 pellet samples collected from four herds belonging to the largest remnant population of Nilgiri tahr in Eravikulam National Park, with 19 microsatellite markers, of which 17 polymorphic markers were selected for further tests. We observed varied levels of polymorphism (2–8 alleles) and heterozygosity (0.0476–0.8421). Probability of identity for individuals was 0.0018 at 10 loci and for siblings was 0.0062 at 13 loci, signifying the usefulness of these markers to study wild herds. Overall, observed and expected heterozygosities were H _o = 0.4280 ± 0.2376 and H _e = 0.4464 ± 0.2265, respectively, and the F _IS value was 0.0138 (p = 0.63). Our results validate the use of cross-species markers in wild populations of Nilgiri tahr to identify individuals and determine genetic diversity, which can be further used to understand population dynamics of this species.     

Molecular phylogeography and paleodistribution modeling of the boreal tree species <Emphasis Type="Italic">Ulmus lamellosa</Emphasis> (T.Wang et S. L. Chang) (Ulmaceae) in China

The uplift of mountains and climatic oscillations are important for understanding of the demographic history and genetic structure of species. We investigated the biogeographic history of the boreal tree species Ulmus lamellosa (Ulmaceae) in China, by using a combined phylogeographic and paleodistribution modeling approach. In this study, 14 populations of endangered U. lamellosa were analyzed by using chloroplast DNA (cpDNA) sequences. A high level of genetic differentiation (Φ _ST = 86.22%) among populations with a significant phylogeographic pattern (N _ST > G _ST, P < 0.05) was found in U. lamellosa. Ten haplotypes were detected by combining chloroplast DNA data, and haplotype 3 (H3) was found to be common and widespread. The intraspecific divergence of all U. lamellosa cpDNA haplotypes (9.27 Ma; 95% HPD 5.17–13.33 Ma) most probably began in the late Miocene. The pairwise difference among haplotypes and neutrality tests (Tajima’s D and Fu’s Fs statistic) indicated that populations of U. lamellosa, except group I, have not experienced recent sudden expansions. Multiple refuge areas were identified across the entire distribution ranges of U. lamellosa. The low level of gene flow (Nm = 0.14) among populations may have resulted from isolation resulting from distance and complex topography during climatic oscillations; this isolation was probably the major process that shaped the present distribution of haplotypes. These results support the hypothesis that U. lamellosa persisted in situ during glaciations and occupied multiple localized glacial refugia, contrary to the hypotheses of large-scale range contraction and long-distance southward migration.     

Genetic structure and diversity in relation to the recently reduced population size of the rare conifer, <Emphasis Type="Italic">Pseudotsuga japonica</Emphasis>, endemic to Japan

Rare species consisting of small populations are subject to random genetic drift, which reduces genetic diversity. Thus, determining the relationship between population size and genetic diversity would provide key information for planning a conservation strategy for rare species. We used six microsatellite markers to investigate seven extant populations of the rare conifer Pseudotsuga japonica, which is endemic to the Kii Peninsula and Shikoku Island regions that are geographically separated by the Kii Channel in southwest Japan. The population differentiation of P. japonica was relatively high (F_ST = 0.101) for a coniferous species, suggesting limited gene flow among populations. As expected, significant regional differentiation (AMOVA; p?<?0.05) indicated genetic divergence across the Kii Channel. A strong positive correlation between census population size and the number of rare alleles (r?=?0.862, p?<?0.05) was found, but correlations with major indices of genetic diversity were not significant (allelic richness: r?=?0.649, p?=?0.104, expected heterozygosity: r?=?0.361, p?=?0.426). The observed order of magnitude of correlation with three genetic diversity indices corresponded with the theoretically expected order of each index’ sensitivity (i.e., the rate of decline per generation) to the bottleneck event. Thus, features that exhibit a faster response, i.e., the number of rare alleles, would have been subject to deleterious effects of the recent decline in population size, which is presumably caused by the development of extensive artificial plantations of other tree species over the last several decades. Finally, we propose a conservation plan for P. japonica based on our findings.     

Genetic diversity and population structure of a drought-tolerant species of <Emphasis Type="Italic">Eucalyptus</Emphasis>, using microsatellite markers

Given the impact of climate change on the availability of water resources, it becomes necessary the use of plant species well suited to planting on dryland sites. Eucalyptus cladocalyx, a native tree of South Australia, is capable of growing under relatively dry environments and saline soils. Two hundred twenty simple sequence repeat (microsatellites) markers, from a consensus linkage map of Eucalyptus, were selected to examine genetic diversity and population structure in a collection of E. cladocalyx introduced to southern Atacama Desert, Chile. A total of 130 microsatellites were successfully amplified, some of which are associated with quantitative traits of interest in Eucalyptus. Genetic analysis revealed a total of 457 alleles, ranging from 2 to 8 alleles per locus. A moderate level of genetic diversity (He = 0.492) and differentiation (FST = 0.086) was found among the populations. Mount Remarkable and Marble Range showed the highest and lowest level of genetic diversity, respectively. The Bayesian clustering analysis revealed three homogeneous genetic groups confirming that the individuals of E. cladocalyx from natural forest are highly and significantly structured. These results provide a novel information for the development of breeding strategies in E. cladocalyx by using marker-assisted selection in regions with low rainfall patterns.     

Genetic analysis of forest species <Emphasis Type="Italic">Eugenia uniflora</Emphasis> L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA) _n and (CA) _n microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeirão Preto, Tambaú and São José do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guaçu river basins, in state of São Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H _E  = 0.83) and a low genetic differentiation (mean F _ST = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.     

Unspecific histological and hematological alterations in anadromous and resident <Emphasis Type="Italic">Salvelinus malma</Emphasis> induced by volcanogenic pollution

Knowledge of larval dispersal and connectivity in coral reef species is crucial for understanding population dynamics, resilience, and evolution of species. Here, we use ten microsatellites and one mitochondrial marker (cytochrome b) to investigate the genetic population structure, genetic diversity, and historical demography of the powder-blue tang Acanthurus leucosternon across more than 1000 km of the scarcely studied Eastern African region. The global AMOVA results based on microsatellites reveal a low but significant F _ST value (F _ST = 0.00252 P < 0.001; D _EST = 0.025 P = 0.0018) for the 336 specimens sampled at ten sample sites, while no significant differentiation could be found in the mitochondrial cytochrome b dataset. On the other hand, pairwise F _ST, PCOA, and hierarchical analysis failed to identify any genetic breaks among the Eastern African populations, supporting the hypothesis of genetic homogeneity. The observed genetic homogeneity among Eastern African sample sites can be explained by the lengthy post-larval stage of A. leucosternon, which can potentiate long-distance dispersal. Tests of neutrality and mismatch distribution signal a population expansion during the mid-Pleistocene period.     

Genetic assessment of <Emphasis Type="Italic">Abies koreana</Emphasis> (Pinaceae), the endangered Korean fir,and conservation implications

To establish a management plan for endangered and rare species, genetic assessment must first be conducted. The genetic characteristics of plant species are affected by demographic history, reproductive strategy, and distributional range as well as anthropological effects. Abies koreana E. H. Wilson (Pinaceae), Korean fir, is endemic to Korea and found only in sub-alpine areas of the southern Korean Peninsula and Jejudo Island. This species has been designated as critically endangered by the International Union for Conservation of Nature due to a continuous decline in its range and population fragmentation. We genotyped 176 individuals from seven natural populations and two afforested populations on the Korean Peninsula using 19 microsatellite loci. STRUCTURE analysis revealed two genetic clusters in natural populations (F _st  = 0.040 and R _st  = 0.040) despite low differentiation. We did not detect a significant reduction in genetic diversity or the signature of a genetic bottleneck despite population fragmentation and small population size. We deduced that this species exhibits a metapopulation structure, with the population on Jirisan Mountain acting as a source of genetic diversity for other local small populations on the Korean Peninsula, through contemporary asymmetric gene flow. However, the majority of afforested individuals on the Korean Peninsula originated from a different gene cluster. Thus, we recommend a conservation strategy that maintains two genetically unique clusters.     

Using genetic monitoring to inform best practice in a captive breeding programme: inbreeding and potential genetic rescue in the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis>

Freshwater pearl mussel (Margaritifera margaritifera) populations are declining in Northern Ireland to the extent that a captive breeding programme was established on the Upper Ballinderry river in 1998. Previous genetic analysis of the hatchery broodstock and their first cohort of offspring showed significant levels of inbreeding (F _IS  = 0.166). The broodstock, which currently numbers ca. 90 individuals, was supplemented with new individual mussels, whilst in 2013, a previously unknown population was discovered on the Lower Ballinderry river. The aim of the present study was to determine whether the rotation of the broodstock has led to a decrease in the levels of inbreeding in the second cohort of juveniles, and to determine whether the new population found in the Lower Ballinderry was genetically distinct from the captive bred population and populations from the Upper Ballinderry, which represent the source of the hatchery broodstock. Genotyping using eight microsatellite markers indicated that levels of inbreeding in the second cohort of captive-bred mussels were high, (F _IS  = 0.629), and were comparable to those sampled from the original cohort and the hatchery broodstock (F _IS  = 0.527 and 0.636 respectively). Bayesian analysis of population structure indicated that the newly discovered Lower Ballinderry population was genetically distinct from the broodstock and its source populations on the Upper Ballinderry. The observed differentiation was primarily due to differences in allele frequencies, and was most likely a result of genetic drift. The occurrence of ten alleles, albeit at low frequency, in the Lower Ballinderry population, including four private alleles, suggests that this new population could be incorporated into the broodstock with the aim of decreasing levels of inbreeding in the future.     

Genetic diversity and population structure of <Emphasis Type="Italic">Melia azedarach</Emphasis> in North-Western Plains of India

Key message

Genetic structure among M. azedarach populations was detected and two subpopulations were present among them. A significant ‘isolation by distance’ was found in M. azedarach population in North-Western Plains of India.

Abstract

Melia azedarach is an important forest tree with pharmaceutical, insecticidal, pesticidal, and commercial significance. It is a good reforestation tree because of its fast growth and drought hardy nature. Genetic variation in a species allows itself to adapt, evolve and respond to environmental stress. It provides the basis for survival of a species and critically influences its evolutionary potential. Assessment of genetic diversity is necessary for improvement and conservation of a species. For this, microsatellite markers are of particular interest given the attributes like co-dominance, reproducibility, hyper variability and abundance throughout the genome. In the present study, we analyzed the genetic diversity and population structure of M. azedarach, an ecologically imperative species growing in the North-Western Plains of India. We developed 43 microsatellite markers, of which 20 were subsequently employed for analysis of diversity and population structure among 33 populations encompassing 318 genotypes representing North-Western Plains of India. A moderate level of diversity (Na = 5.1, Ho = 0.506, He = 0.712, I = 1.386) was assessed. The highest value of ΔK estimated using STRUCTURE indicated 2 subpopulations (K = 2). AMOVA exhibited 73 % variation within populations and 12 % variation was found among regions. Significant positive correlation between geographical and genetic distance was found (Rxy = 0.365, P = 0.010). The present study lays a foundation on a better understanding of genetic dynamics of the species and reveals its diversity and population structure in North-Western Plains of India.

     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.