Croatian Populations Research Articles

Nuclear ITS and AFLPs provide surprising implications for the taxonomy of Tephroseris longifolia agg. and the endemic status of T. longifolia subsp. moravica

The proportion of endemic taxa is a frequently used measure for assesment of biodiversity hotspots within the Carpathians. For confirming of an endemic status of a taxon, a thorough evaluation using modern tools of plant systematics is desirable. Tephroseris longifolia subsp. moravica has been considered to be a Western Carpathian endemic, but a previous study of morphological and genome size differentiation within the T. longifolia agg. showed that its endemic position is controversial. Therefore, in this study we aimed to detect genetic variability and evolutionary relationships within the T. longifolia agg. using nuclear ITS sequences and highly variable AFLPs on 38 populations covering the distribution range of the aggregate. Additionally, we analysed 22 populations of other putatively related species (T. aurantiaca, T. capitata, T. crispa, T. integrifolia and T. papposa). Genetic analyses proved that T. longifolia agg. is genetically well differentiated, but the results contradict the current taxonomic concept. Tephroseris longifolia aggregate, as defined here, comprises T. longifolia, T. crispa, T. pseudocrispa, T. tenuifolia and T. italica. Within T. longifolia, two subspecies can be recognised, namely T. l. subsp. longifolia and T. l. subsp. moravica. Furthermore, AFLPs confirm the close relationship of Pannonian and one Croatian populations to T. l. subsp. moravica. Therefore, we assume that T. l. subsp. moravica should not be considered a Western Carpathian endemic and most probably migrated to the Western Carpathian area during the post-glacial period.

Phytochemical and Micromorphological Characterization of Croatian Populations of Erodium cicutarium

The aim of this study included the phytochemical screening of extracts and essential oil of Erodium cicutarium (L.) L’Hér. ex Aiton (Geraniaceae), a native traditional medicinal plant from Croatia, as well as a highly detailed micromorphological characterization of its aerial parts. The contents of total polyphenols and tannins (TP and T), total flavonoids (TF), and total hydroxycinnamic derivatives (THD) in plant extracts were quantified in plant material from 4 localities (Plitvice, Podvinje, Buzin, and Trešnjevka). The contents of TP, T, TF, and THD significantly varied depending on the collection site, and were in the range of 4.78%-12.85% (TP), 3.23%-5.80% (T), 0.42%-1.09% (TF), and 1.08%-2.59% (THD) of dry weight of plant material, with the Plitvice collection having the highest polyphenol content. GC-MS analysis of the essential oils showed a similarity in composition of the major compounds from all investigated populations. Fifty compounds were identified in all 4 investigated oils (90.4%-96.7% of total oil) and classified into 7 structural classes, with hydrocarbons (59.8%-65.7%) as the main class of constituents. Two types of trichomes, nonglandular and glandular, were observed on the calyces, leaves, and stems, including 3 capitate trichome types. The bioactive substances and micromorphological characterization of Croatian populations of E. cicutarium were investigated for the first time. In general, the scanning electron microscopy imaging of Erodium cicitarium trichomes has not been reported before.

Genetic Diversity of Cryphonectria hypovirus 1, a Biocontrol Agent of Chestnut Blight, in Croatia and Slovenia.

Transmissible hypovirulence associated with Cryphonectria hypovirus 1 (CHV1) has been used for biological control of chestnut blight, devastating disease of chestnut caused by the fungus Cryphonectria parasitica. The main aims of this study were to provide molecular characterization of CHV1 from Croatia and Slovenia and to reveal its genetic variability, phylogeny, and diversification of populations. Fifty-one CHV1 haplotypes were detected among 54 partially sequenced CHV1 isolates, all belonging to Italian subtype (I). Diversity was mainly generated by point mutations while evidence of recombination was not found. The level of conservation over analyzed parts of ORF-A proteins p29 and p40 varied, but functional sites were highly conserved. Phylogenetic analysis revealed close relatedness and intermixing of Croatian and Slovenian CHV1 populations. Our CHV1 isolates were also related to Swiss and Bosnian hypoviruses supporting previously suggested course of CHV1 invasion in Europe. Overall, this study indicates that phylogeny of CHV1 subtype I in Europe is complex and characterized with frequent point mutations resulting in many closely related variants of the virus. Possible association between variations within CHV1 ORF-A and growth of the hypovirulent fungal isolates is tested and presented.

Essential Oil Composition of Different Plant Parts from Croatian Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (Asteraceae).

Essential oil compositions of fresh leaves, flower stems and rhizomes obtained by hydrodistillation from different Croatian populations of Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (four of each species) were investigated using gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. Altogether, 118 constituents were identified, accounting for 81.19-96.81 % of total oil composition. All essential oils were characterized by oxygenated sesquiterpenes, with distinct compounds recorded for both investigated species. Clear separation between the two species was confirmed by principal component analysis and hierarchical cluster analysis. This is the first study that recorded intraspecific variations of essential oil constituents from P. albus and P. hybridus. Obtained results could contribute to the understanding of their medicinal and nutritional value.

Genome-wide meta-analysis identifies novel loci associated with parathyroid hormone level

BackgroundParathyroid hormone (PTH) is one of the principal regulators of calcium homeostasis. Although serum PTH level is mostly accounted by genetic factors, genetic background underlying PTH level is insufficiently known. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels.MethodsWe performed GWAS meta-analysis within two genetically isolated Croatian populations followed by replication analysis in a Croatian mainland population and we also combined results across all three analyzed populations. The analyses included 2596 individuals. A total of 7,411,206 variants, imputed using the 1000 Genomes reference panel, were analysed for the association. In addition, a sex-specific GWAS meta-analyses were performed.ResultsPolymorphisms with the lowest P-values were located on chromosome 4 approximately 84 kb of the 5′ of RASGEF1B gene. The most significant SNP was rs11099476 (P = 1.15 × 10−8). Sex-specific analysis identified genome-wide significant association of the variant rs77178854, located within DPP10 gene in females only (P = 2.21 × 10− 9). There were no genome-wide significant findings in the meta-analysis of males.ConclusionsWe identified two biologically plausible novel loci associated with PTH levels, providing us with further insights into the genetics of this complex trait.

SOCIOECONOMIC INFLUENCE OF TOBACCO USE AND DENTAL CARIES EXPERIENCE IN DIFFERENT MALE CROATIAN POPULATIONS.

SUMMARY – The purpose was to evaluate dental caries experience in different male subpopulations of Croatian Army recruits and dental students, and subsequently, smoking habits related to the level of education and place of residence. Croatian army recruits (n=248; mean age 20.2) and male dental students (n=56; mean age 21.5) were evaluated according to DMFT and FST indices, divided according to age and place of residence, and interviewed about their dietary habits and smoking of tobacco. In the subpopulation of recruits, the median value of DMFT was 6 and of FST index 25. A statistically significant difference was recorded between DT and FST index (p<0.05) according to dietary role of carbohydrates reflected in caries development. The number of recruits with finished elementary school coming from a rural area who smoked (in total 57.66% of smokers) was significantly larger (p=0.0041). In dental students, the median value of DMFT was 5, with statistical significance in comparison with recruits (p=0.03). There was a difference in FST index (median 28) (p<0.0001). Students were mostly nonsmokers (71.43%) and had urban residence (p<0.0001). FST index was a more specific indicator in the socioeconomically heterogeneous sample of recruits (more subjects coming from rural areas and with a lower level of education) than in the sample of dental students (more subjects coming from urban areas).

Genetic variation and forensic characteristic analysis of 25 STRs of a novel fluorescence co-amplification system in Chinese Southern Shaanxi Han population.

We analyzed the genetic polymorphisms of 15 autosomal and 10 Y-chromosomal STR loci in 214 individuals of Han population from Southern Shaanxi of China and studied the genetic relationships between Southern Shaanxi Han and other populations. We observed a total of 150 alleles at 15 autosomal STR loci with the corresponding allelic frequencies ranging from 0.0023 to 0.5210, and the combined power of discrimination and exclusion for the 15 autosomal STR loci were 0.99999999999999998866 and 0.999998491, respectively. For the 10 Y-STR loci, totally 100 different haplotypes were obtained, of which 94 were unique. The discriminatory capacity and haplotype diversity values of the 10 Y-STR loci were 0.9259 and 0.998269, respectively. The results demonstrated high genetic diversities of the 25 STR loci in the population for forensic applications. We constructed neighbor-joining tree and conducted principal component analysis based on 15 autosomal STR loci and conducted multidimensional scaling analysis and constructed neighbor-joining tree based on 10 Y-STR loci. The results of population genetic analyses based on both autosomal and Y-chromosome STRs indicated that the studied Southern Shaanxi Han population had relatively closer genetic relationship with Eastern Han population, and distant relationships with Croatian, Serbian and Moroccan populations.

Like a Bridge Over Troubled Adriatic Water: The Complex Relationship between Italian and Yugoslavian Sporting Diplomacy (1945–1954)

The fall of the Fascist regime and the creation of the FPRY did not end the pre-existing ethnic strains between the Slovenian–Croatian and Italian populations, nor did it temper the geopolitical ambitions of Yugoslavia and Italy over the Adriatic coast. Furthermore, the political choices of Belgrade and Rome caused, especially up to 1948, growing differences from an ideological standpoint and influenced the sporting field. The aim of this paper is to analyze the evolution of the sporting–diplomatic relationship between Italy and Yugoslavia, paying special attention to the ‘Trieste case’. Unsurprisingly, before 1948, there were no bilateral sporting exchanges; however, the two countries were particularly committed to demonstrate, including in the sporting field, that Trieste belonged to them. Belgrade could take advantage of the presence of a strong communist Italian minority, who created a sport association for the Free Territory of Trieste whose clubs played in the Yugoslavian championships. In a similar way, the Italian Olympic Committee constantly used sport to affirm the ‘Italianness’ of Trieste. After the Tito–Stalin split, the ‘sporting fight’ to ‘occupy’ Trieste lost intensity as Belgrade began to show more interest in reopening the (sporting) relationships with the West. By looking at the evolution of the bilateral relations between Italy and Yugoslavia, it will be argued that the timing of this sporting rapprochement was strongly connected with the political diplomacy between Belgrade and Rome.

Allele frequencies of 15 STR loci in Bosnian and Herzegovinian population.

AimTo determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations.MethodsGenomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlex® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations.ResultsThe highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus.ConclusionUpon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies.

Molecular and morphometric characterisation of the invasive signal crayfish populations in Croatia

The signal crayfish Pacifastacus leniusculus (Dana, 1852) is a native species to North America. It was introduced to Europe and Japan where it rapidly spread as an invasive species. In Croatia, it is recorded in the rivers Mura and Drava, where it spread downstream from Slovenia, and in the Korana River, where it has been recently illegally introduced. In the invaded areas, signal crayfish outcompetes native crayfish species. Since the knowledge on the genetic diversity of this invasive species is limited, microsatellite markers and sequences of mitochondrial gene for 16S rRNA were analysed to explore the genetic relations between the two Croatian populations (Mura and Korana rivers) as well as their relation to other already studied European populations. Moreover, 16S rRNA sequence fragments of Croatian samples were compared with those from the native range in the west North America. Morphometric characteristics were also studied to determine if there are significant differences between studied populations and if these are concordant with the genetic analyses results. Also, morphometric data were used to assign Croatian signal crayfish into subspecies classification according to Miller’s discriminant function formula, and to compare claw surface area among Croatian, Japanese and North American populations. Based on the results of morphometric characteristics Croatian samples showed Pacifastacus leniusculus leniusculus -like morphology. Phylogenetic reconstruction based on 16S rRNA, positioned Croatian samples into P. leniusculus sensu Larson et al. (2012). Results on microsatellite markers showed that the genetic diversity of P. leniusculus in the Mura River population is slightly higher compared to the Korana River population, but these two populations do not form separated genetic clusters. This study contributes to the knowledge on genetic variability and morphometric characteristics of signal crayfish in Europe and further understanding of its success as an invasive species.

The lack of association between angiotensin-converting enzyme gene insertion/deletion polymorphism and nicotine dependence in multiple sclerosis.

ObjectiveBlood‐borne angiotensin II is generated from angiotensinogen via cleavage by renin and angiotensin‐converting enzyme (ACE), an enzymatic cascade known as the renin–angiotensin system (RAS). Several lines of evidence indicate that ACE, beyond its classical role of mediating blood pressure regulation, might contribute to the etiology of substance addictions by influencing dopaminergic signaling. A functional insertion/deletion (I/D) polymorphism of the ACE gene was associated with risk for being a smoker among individuals with depression and with smoking severity in studies comprising patients with depression and healthy controls. Several reports have described significantly increased ACE activity in cerebrospinal fluid and serum among MS patients. Furthermore, in our previous work with MS patients from Croatian and Slovenian populations, we demonstrated that the ACE‐I/D polymorphism contributes to an elevated MS risk among male patients. Here we investigated whether the ACE‐I/D polymorphism might influence smoking behavior among patients with MS.Patients and MethodsGenotyping was performed in 521 patients (males/females: 139/382) using polymerase chain reaction.ResultsWe revealed no significant differences in ACE genotype and allele frequencies between smokers and nonsmokers and no significant association between the ACE‐I/D polymorphism and either pack‐year smoking history or number of cigarettes smoked daily (p > .05, respectively).ConclusionThe ACE‐I/D polymorphism does not contribute either to risk for nicotine dependence or to smoking severity among MS patients. In the context of reports on the ACE‐I/D polymorphism and nicotine dependence among healthy controls and patients with depression, we may speculate that the mechanism by which this polymorphism influences nicotine dependence risk differs in MS compared to depression, although not compared to a healthy population. In addition to angiotensin II, other potential ACE substrates, such as substance P and neurotensin, which also influence dopaminergic neurotransmission (and are proposed to be associated with MS), may deserve study in future.

Goniolimon dalmaticum Rchb. f. and G. tataricum (L.) Boiss. (Plumbaginaceae) in the Croatian Flora and their Distribution in the Balkan Peninsula

Abstract Goniolimon tataricum (L.) Boiss. is recognized as a new species for the Croatian flora. Besides G. dalmaticum Rchb. f., G. tataricum represents the second species of this genus in Croatia. To confirm the identity of G. tataricum as a new species, morphometric study and multivariate statistical analysis were performed on the data from four Croatian populations of G. dalmaticum and G. tataricum. Based on results of morphometric analysis, an identification key for these two species is provided. Data about distribution in the Balkan Peninsula, habitat and ecology of the newly-recorded species are also presented. Typification of G. dalmaticum was performed for the first time.

Spatio-temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae).

Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers exhibiting a highly specialised pollination system. The sole pollinators are kleptoparasitic flies in search of food. This study investigates these pollinators on a spatio-temporal scale and the impact of weather conditions on their availability. Two potential strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and an increased floral life span, were tested. A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded in two populations according to pollinator availability. Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollinator compositions varied among populations. Weather conditions 15-30days before pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity depending on pollinator availability. A.rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of pollinators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during periods of pollinator limitation.

Geographical location is a key component to effective breeding of clary sage (Salvia sclarea) for essential oil composition

Wild populations of Croatian clary sage (Salvia sclarea L.) were examined for variability to determine cultivation suitability in Croatia and Israel for breeding purposes. Phenotypic variability (coefficient of variation; %) was recorded for inflorescence weight (39.6%), inflorescence yield (52.8%), and essential oil yield (67.6%) when grown in Croatia. Associations were identified between inflorescence yield and essential oil yield (r = 0.9; P &lt; 0.0001), inflorescence weight and inflorescence yield (r = 0.8; P &lt; 0.0001), and inflorescence weight and inflorescence length (r = 0.6; P = 0.0056), suggesting that populations with elongated inflorescence are indirectly associated with higher essential oil yield. In Israel, the populations reached full bloom between the end of May and early June, corresponding on average to 397.5 days post planting. Linalyl acetate, linalool, α–terpineol, sclareol, and geranyl acetate were the leading essential oil components in both Croatia and Israel. The principal compounds in the oil were linalyl acetate (48.5%) and linalool (17.7%), signifying that the Croatian populations were of the linalool chemotype. A two-way ANOVA indicated an interaction between growing location (Croatia vs. Israel) and population for linalool (P = 0.02), α–terpineol (P = 0.007), and linalyl acetate (P = 0.09); evidence of an environmental effect on essential oil composition. The variation observed suggested that the wild population of clary sage in Croatia had the genetic heterogeneity essential for breeding. Nevertheless, the differences in essential oil composition between Croatia and Israel suggest that breeding efforts should be separately focused for each agriculture production system.

A Case-control Study in an Orcadian Population Investigating the Relationship between Human Plasma N-glycans and Metabolic Syndrome

Background: Alterations in glycosylation patterns have long been known to reflect changes in cell metabolism. In this study, we investigated the relationship between human N-glycan profiles and metabolic syndrome. Method: Between 2005 and 2011, 2,155 individuals from the Orkney Islands (UK) were recruited and biological material, alongside phenotypic measures were collected. Individual N-glycan profiles were measured in plasma using weak anion exchange high performance liquid chromatography and calibrated hydrophilic interaction liquid chromatography. Pre-specified criteria were used to identify 564 cases with metabolic syndrome and 1475 controls. We applied logistic regression to test for association between this binary outcome against measured plasma N-glycans. We also assessed the correlation between N-glycan traits and individual components of metabolic syndrome and compared this to results found in similar analyses based in Chinese and Croatian populations. Results: 21 N-glycan traits were found to be associated with either an increased or a decreased likelihood of participants having metabolic syndrome, including monosialylated plasma N-glycans (OR of 1.49 (95%CI 1.33, 1.67), q=1.26E-12) and core fucosylated plasma N-glycans (OR of 0.81(95% CI 0.72-0.90), q=7.75E-4). Notably, consistent results in both sections of this analysis demonstrated the protective association of higher levels of core fucosylated N-glycans. Conclusion: Our results demonstrate that metabolic syndrome is associated with an alteration in plasma N-glycosylation patterns. The metabolic role of core fucosylated N-glycans is of particular interest for future study.

Ecological morphology of the sugar beet weevil Croatian populations: Evaluating the role of environmental conditions on body shape

Different rearing environments produce dissimilar stress level in insects, which can be reflected on the ability of an individual to overcome these pressures and spread further. Sugar beet weevil, Bothynoderes punctivnetris Germar. (Coleoptera: Curculionidae) is one of the most harmful pests of sugar beet, infesting agricultural fields in different parts of Croatia. It is highly invasive being able to inhabit diverse environments across the country. The aim of this study was to assess the association between different environmental and rearing conditions and the morphological variation observed in the sugar beet weevil body shape. The present results showed significantly different eco-morphs related to different environmental conditions, thus suggesting that the observed ecomorphological changes could be associated to the ability of this pest to spread further.

Association of the FAM46A gene VNTRs and BAG6 rs3117582 SNP with non small cell lung cancer (NSCLC) in Croatian and Norwegian populations.

We analyzed for associations between a variable number of tandem repeat (VNTR) polymorphism in the Family with sequence similarity 46, member A (FAM46A) gene and a single nucleotide polymorphism (rs3117582) in the BCL2-Associated Athanogene 6 (BAG6) with non small cell lung cancer in Croatian and Norwegian subjects. A total of 503 (262 Croatian and 241Norwegian) non small cell lung cancer patients and 897 controls (568 Croatian and 329 Norwegian) were analyzed. We found that the frequency of allele b (three VNTR repeats) of FAM46A gene was significantly increased in the patients compared to the healthy controls in the Croatian and the combined Croatian and Norwegian subjects. Genotype frequencies of cd (four and five VNTR repeats) and cc (four VNTR repeats homozygote) of the FAM46A gene were significantly decreased in the patients compared to the healthy controls in the Croatian and Norwegian subjects, respectively. Logistic regression analyses revealed FAM46A genotype cc to be an independent predictive factor for non small cell lung cancer risk in the Norwegian subjects after adjustment for age, gender and smoking status. This is the first study to suggest an association between the FAM46A gene VNTR polymorphisms and non small cell lung cancer. We found also that BAG6 rs3117582 SNP was associated with non small cell lung cancer in the Norwegian subjects and the combined Croatian-Norwegian subjects corroborating the earlier finding that BAG6 rs3117582 SNP was associated with lung cancer in Europeans. Logistic regression analyses revealed that genotypes and alleles of BAG6 were independent predictive factor for non small cell lung cancer risk in the Norwegian and combined Croatian-Norwegian subjects, after adjustment for age and gender.

Analyzing large-scale samples confirms the association between rs16892766 polymorphism and colorectal cancer susceptibility.

Colorectal cancer (CRC) is a common complex disease caused by the combination of genetic variants and environmental factors. Genome-wide association studies (GWAS) have been performed and reported some novel CRC susceptibility variants. The rs16892766 (8q23.3) polymorphism was first identified to be significantly associated with CRC in European ancestry. The following studies investigated this association in Chinese, Japanese, Romanian, Swedish, African American, European American, and Croatian populations. These studies reported consistent and inconsistent results. Here, we reevaluated this association using the relatively large-scale samples from 13 studies (N = 59737, 26237 cases and 33500 controls) using a meta-analysis by searching the PubMed, Google Scholar and CRCgene databases. We observed no significant heterogeneity among the included studies. Our results showed significant association between rs16892766 polymorphism and CRC (P = 1.33E-35, OR = 1.23, 95% CI 1.20-1.27). Collectively, our analysis further supports previous findings that the rs16892766 polymorphism is significantly associated with CRC susceptibility. We believe that our findings will be very useful for future genetic studies on CRC.

Genetic Monitoring of Western Corn Rootworm (Coleoptera: Chrysomelidae) Populations on a Microgeographic Scale

Microsatellite and mitochondrial DNA genetic monitoring of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was undertaken in Croatia and Serbia from 1996 to 2011 and in the United States in 2011. The seven U.S. populations displayed the greatest allelic diversity. In Europe, the highest number of alleles was found in Rugvica, Croatia, and Surčin, Serbia, the two sites closest to international airports. The highest number of mitochondrial (mt) DNA haplotypes was recorded from Croatia in 1996. From 2009 to 2011, haplotype diversity declined, and Croatia and Serbia had a single fixed haplotype. U.S. continuous maize locations had one haplotype, while three haplotypes were found at crop-rotated locations. Minimal temporal genetic differentiation was found within and between populations in Europe and the United States. Bayesian cluster analysis identified two genetic clusters that grouped western corn rootworm from Croatia and Serbia separately from U.S. populations; however, these clusters were not neat, and numerous U.S. individuals had both European and U.S. ancestry, suggesting bidirectional gene flow. Bottlenecks were identified within most Croatian populations sampled in 1996, only two populations in 2009, and in all populations in 2011. Bottlenecks were not identified from Serbia from 1996 to 2011 or from the United States in 2011. As suspected Serbia was identified as the geographic source of western corn rootworm in Croatia. The temporal genetic monitoring undertaken allowed a deeper understanding of the population genetics of western corn rootworm in Croatia, neighboring Serbia, and its geographic source in the United States. The data obtained can be used to inform western corn rootworm pest management strategies in Croatia and Europe.

Intra- and interpopulation relationships and taxonomic status of Pinus nigra Arnold in Croatia according to morphology and anatomy of needles

Intra- and interpopulational relationships and taxonomic status of six natural black pine populations (&lt;em&gt;Pinus nigra&lt;/em&gt; Arnold) from Croatia were analyzed. Special attention in this research was paid to the establishment of taxonomic status and distribution of stenoendemic Dalmatian black pine (&lt;em&gt;Pinus nigra&lt;/em&gt; Arnold subsp. &lt;em&gt;dalmatica&lt;/em&gt; (Vis.) Franco). Three morphological and twenty-one anatomical traits of needles were used as well as analysis by the methods of multivariate statistics. Most of the researched individuals showed strong population affiliation, whereas a clinal transition of morphological and anatomical traits of needles was determined among the populations. The opinion of the existence of three subspecies of black pine on the territory of Republic of Croatia (&lt;em&gt;Pinus nigra&lt;/em&gt; Arnold subsp. &lt;em&gt;nigra&lt;/em&gt;, &lt;em&gt;Pinus nigra&lt;/em&gt; Arnold subsp. &lt;em&gt;dalmatica&lt;/em&gt; (Vis.) Franco and &lt;em&gt;Pinus nigra&lt;/em&gt; Arnold subsp. &lt;em&gt;illyrica&lt;/em&gt; (Vid.) Fukarek) was confirmed. For each of the three subspecies new ranges were represented. Four Croatian populations were included in the stenoendemic subspecies of the Dalmatian black pine (Brač, Hvar, Grobnik and Pelješac), and a new distribution, broken in two, was suggested. Since the Dalmatian black pine has been reduced to several populations only, and there are some signs of continuous diminishing of its range, this research points out the need of making an action plan to preserve all natural populations of this subspecies.