ABSTRACT Using genetic data, we confirm for the first time the presence of the Tanzanian helmeted terrapin Pelomedusa kobe in Akagera National Park, Rwanda, extending their known distribution. Our study provides the first natural history observations, population and morphological data for this species from a wild population. As species in the genus Pelomedusa are morphologically difficult to distinguish, we gain the first insights into intraspecific and interspecific patterns of size and shape variation of plastron morphology between two morphologically‐similar species living in Eastern Africa, P. kobe and P. subrufa , using geometric morphometrics. Species affiliation, rather than sex, is the dominant factor influencing size and shape variation. P. kobe exhibits female‐based sexual size dimorphism (SSD), while P. subrufa shows a tendency toward male‐based SSD. Furthermore, sexual shape dimorphism (SShD) was observed in P. kobe but not in P. subrufa . Our results provide the first integrative data (genetic, morphological and natural history) on P. kobe , however further information on their distribution in the park is needed for effective conservation of this data‐deficient species. Our findings lay the groundwork for future ecological studies on P. kobe , potentially revealing unique adaptations and conservation needs previously overlooked due to its cryptic nature.

Although a significant unexploited potential of the geometric morphometrics in taxonomy and systematics has been documented, only a few studies so far have focused on the utility of this method in the detection of population genetic structure in turtles. For the first time, we employed geometric morphometrics to explicitly analyse whether populations possessing divergent haplotypes evolved different patterns of plastral shape in any Testudo species using the Greek populations of Hermann's tortoise as a case study. To molecularly characterize the Greek populations of Testudo hermanni boettgeri used for plastron shape analyses, 12s rRNA gene of tortoises sampled from seven localities in Greece was sequenced. Three divergent haplotypes were observed, regionally corresponding to the north-western (HI), south-western (HII) and Peloponnese (HIII) areas of Greece. In both females and males, haplotypes HI and HII showed the lack of plastral shape divergence, but were both statistically significantly different relative to HIII found in Peloponnese. The Peloponnesian populations (HIII) had longer bridges and the wider and shorter anterior part of the plastron, relative to populations from the mainland Greece (HI and HII). Hence, both morphological and molecular markers suggested Peloponnese as a distinct lineage in relation to mainland populations. Finally, we consider geometric morphometrics as an efficient method to detect and characterize subtle interpopulation differences. Findings in this study have conservation applications as well, implying the presence of cryptic genetic and taxonomic diversity, and, thus, filling the crucial gap in the assessment of the conservation status of T. hermanni.

The published cytb mtDNA sequences from a previous phylogeographic study of Testudo hermanni and de novo genetic and phenotypic data of samples from the Pcinja River Valley (Serbia, Central Balkans) and Trebinje (Bosnia and Herzegovina) were used to: (i) genetically characterize samples from Pcinja, a region of great importance from the conservation standpoint due to its taxonomic diversity and biogeographical history, and Trebinje, a type locality of a disputed taxon "Testudo hercegoviensis" Werner, 1899; (ii) to study the link between gene genealogy and geographic distribution by implementing spatial genetic analyses, and (iii) to assess whether the distribution of carapace shape variation is in accordance with genetic clusters. The samples from Pcinja and Trebinje possessed divergent haplotypes that corresponded to east (HI) and west (HV) genetic clusters, respectively. Geometric morphometrics was found to be a suitable method to distinguish divergent genetic clusters, arguing for the reassessment of a subspecific ranking within the currently recognized eastern Hermann's subspecies, T. h. boettgeri.

Ecological and evolutionary factors, together with abiotic conditions, affect biogeo-graphic patterns of genetic entities. The spatial and temporal variability of chromosomal inversions of Drosophila subobscura suggests that this species can serve as a good model for studying the effects of environmental change on the genetic structure of natural populations. A comprehensive meta-analysis of the association of environmental and climatic variables with inversion diversity patterns was performed on 20 D. subobscura populations from the central part of the Balkan Peninsula. Environmental data consisted of 3 sets of variables related to temperature and precipitation, extracted from 2 climatic databases, averaged over a 3 month period, and using biological instead of calendar dates of sampling. Arrangement frequency patterns are likely driven by a synergistic effect of factors related to temperature and precipitation. The frequencies of standard chromosomal arrangements tend to co-vary positively with precipitation, whereas parameters related to temperature appear to favor higher frequencies of the inverted and more complex chromosomal arrangements. A complex relationship among local environmental variables is evident from the results and reflects the probable effect of an altitudinal shift; the altitudinal gradient of inversions is different from their latitudinal gradient. The pattern of inversions is generally not associated with environmental variables, and a particular inversion cannot be a predictive genetic marker of global climate change. Populations in different habitats are subjected to habitat-specific selection regimes, while demographic factors and population history also affect the genetic variability pattern observed.

Pores and sensilla on ostracod shell have often been used in studies of ontogeny, taxonomy, and phylogeny of the group. However, an analysis of sexual dimorphism and variation between valves in the number and distribution of pores is lacking. Also, such studies have never been done on a widely distributed, morphologically variable, and weakly ornamented freshwater ostracod. Here, we survey pores in one such species, Physocypria kraepelini. We choose 27 homologous pores as landmarks for 2D-geometric morphometric analysis, with the aim to assess intersexual and between valves variation in size and shape relative to the Fourier outline analysis. This species has only simple (Type A) pores with and without a lip, and each pore carries an undivided sensory seta. Our results show that the total number of pores varies (from 270 to 296), but this is not associated with a specific valve. Males carry fewer pores than females, however no sex specific pores are found. Small intrapopulation divergence of the Cyt b molecular marker (1%) indicates that morphological variability is not species related. We found that P. kraepelini exhibits directional asymmetry of size and shape, sexual size dimorphism (SSD) but lacks sexual shape dimorphism (SShD). Two geometric morphometrics methods were congruent in the estimation of SSD, SShD, and directional asymmetry of shape but differ in the statistical evaluation of directional asymmetry of size. Contrary to other animal groups, our study suggests that ostracods have more pronounced directional asymmetry of shape compared to directional asymmetry of size.

Discovery of cryptic species using molecular tools has become common in many animal groups but it is rarely accompanied by morphological revision, creating ongoing problems in taxonomy and conservation. In copepods, cryptic species have been discovered in most groups where fast-evolving molecular markers were employed. In this study at Yeelirrie in Western Australia we investigate a subterranean species complex belonging to the harpacticoid genus Schizopera Sars, 1905, using both the barcoding mitochondrial COI gene and landmark-based two-dimensional geometric morphometrics. Integumental organs (sensilla and pores) are used as landmarks for the first time in any crustacean group. Complete congruence between DNA-based species delimitation and relative position of integumental organs in two independent morphological structures suggests the existence of three distinct evolutionary units. We describe two of them as new species, employing a condensed taxonomic format appropriate for cryptic species. We argue that many supposedly cryptic species might not be cryptic if researchers focus on analyzing morphological structures with multivariate tools that explicitly take into account geometry of the phenotype. A perceived supremacy of molecular methods in detecting cryptic species is in our view a consequence of disparity of investment and unexploited recent advancements in morphometrics among taxonomists. Our study shows that morphometric data alone could be used to find diagnostic morphological traits and gives hope to anyone studying small animals with a hard integument or shell, especially opening the door to assessing fossil diversity and rich museum collections. We expect that simultaneous use of molecular tools with geometry-oriented morphometrics may yield faster formal description of species. Decrypted species in this study are a good example for urgency of formal descriptions, as they display short-range endemism in small groundwater calcrete aquifers in a paleochannel, where their conservation may be threatened by proposed mining.

The Mediterranean basin is a region of species richness, rarity and endemism, and is thus an area of significant conservation importance. Conservation of insect biodiversity benefits pollination services in the fragmented Mediterranean landscape. However, the question of the distribution patterns of an important long-distance pollen disperser, the hoverfly Eristalis tenax (Diptera, Syrphidae), remains open. Therefore, we explored the spatial distribution of genetic and phenotypic diversity of this species across the Central-Eastern Mediterranean. Connectivity between continental and island populations of E. tenax was studied by integrating molecular (allozyme loci and mitochondrial DNA sequences of the cytochrome c oxidase subunit I gene) and phenotypic data (wing geometric morphometrics). Allozyme (FST values, STRUCTURE analysis, analysis of molecular variance) and phenotypic data (wing shape) and landscape genetics (Alleles in Space analysis) suggest that E. tenax populations within the Mediterranean are largely connected, and that there is an absence of large-scale geographic structuring. Intraspecific variability was only 1.54% among samples, and the Mediterranean populations showed an almost complete lack of COI mtDNA haplotype diversity. Thus, our results suggest that E. tenax populations are well mixed, and that a considerable amount of gene flow takes place, even among populations that are a great distance apart. As E. tenax's ecological amplitude is wide, and the species is therefore widespread in both natural and man-made landscapes, it probably maintains a high level of population similarity by its large population sizes (as revealed by the θ parameter) and constant intermixing among populations. From an applied point of view, large-scale species intermixing enables pollen spread across distant island plant populations, especially those threatened by extinction.

Because of its importance as a pollinator and its potential economic usefulness for the biodegradation of organic animal waste, the genetic and phenotypic diversity of the drone fly, Eristalis tenax L. (Diptera: Syrphidae), was studied in both wild and captive populations from southeastern Europe. Wild specimens from a natural protected habitat (with low human impact), field crop habitat (semisynanthropic condition), and intensive pig farming habitat (synanthropic condition) were compared with a laboratory colony reared on artificial media. An integrative approach was applied based on allozyme loci, cytochrome c oxidase I mitochondrial DNA, wing traits (size and shape), and abdominal color patterns. Our results indicate that the fourth and eighth generations of the laboratory colony show a severe lack of genetic diversity compared with natural populations. Reduced genetic diversity in subsequent generations (F4 and F8) of the laboratory colony was found to be linked with phenotypic divergence. Loss of genetic variability associated with phenotypic differentiation in laboratory samples suggests a founder effect, followed by stochastic genetic processes and inbreeding. Hence, our results have implications for captive bred Eristalis flies, which have been used in crop pollination and biodegradation of organic waste under synanthropic conditions.

Musca domestica L. (Diptera: Muscidae) is a vector of a range variety of pathogens infecting humans and animals. During a year, housefly experiences serial population bottlenecks resulted in reduction of genetic diversity. Population structure has also been subjected to different selection regimes created by insect control programs and pest management. Both environmental and genetic disturbances can affect developmental stability, which is often reflected in morphological traits as asymmetry. Since developmental stability is of great adaptive importance, the aim of this study was to examine fluctuating asymmetry (FA), as a measure of developmental instability, in both wild populations and laboratory colonies of M. domestica. The amount and pattern of wing shape FA was compared among samples within each of two groups (laboratory and wild) and between groups. Firstly, the amount of FA does not differ significantly among samples within the group and neither does it differ between groups. Regarding the mean shape of FA, contrary to non-significant difference within the wild population group and among some colonies, the significant difference between groups was found. These results suggest that the laboratory colonies and wild samples differ in buffering mechanisms to perturbations during development. Hence, inbreeding and stochastic processes, mechanisms dominating in the laboratory-bred samples contributed to significant changes in FA of wing shape. Secondly, general patterns of left-right displacements of landmarks across both studied sample groups are consistent. Observed consistent direction of FA implies high degrees of wing integration. Thus, our findings shed light on developmental buffering processes important for population persistence in the environmental change and genetic stress influence on M. domestica.

A study of population connectivity of the migratory insect species, such as dronefly Eristalis tenax (Diptera, Syrphidae), has an essential importance in understanding the relative influence of the evolutionary forces and environmental features that interact in the spatial distribution of molecular and morphological diversity. However, specific study aiming to understand spatial genetic structure of dronefly populations and its migratory potential is lacking. Hence, we studied a spatial pattern of genetic and phenotypic variation of seven European populations of E. tenax incorporating landscape genetic methods using allozyme data, wing size and shape and abdominal colour pattern. Based on the observed lack of genotypic structuring, we suggested that there has been sufficient long-distance gene flow to effectively homogenize population structuring at a broader geographical scale. Wing shape similarity among populations and an overlap of abdominal colour variation showed no clear clustering related to geography, which is in congruence with genetic data. However, genetic (F-ST values) and phenotypic (wing size) data and landscape genetics indicated subdivision between the Balkan populations (four Serbian samples) and populations from Central (Germany and Switzerland) and Northern (Finland) Europe. These findings indicated a potential connection between the Central and Northern Europe supporting the Central European origin of the flies caught in Finland. Thus, by performing spatial analysis and combining genetic-morphological approach, we shed light on the movement pattern in complex landscapes and thus provided the necessary guidelines to a broad-scale analysis of this widespread generalist pollinator.

Correspondence: Sonja Djordjevic (sonjadj@bio.bg.ac.rs) We processed 40 morphological characteristics of 676 adult Hermann’s tortoises (Testudo hermanni) (310 males and 366 females) from four localities in the central parts of the Balkan Peninsula. Analyses of variance and covariance showed significant differences between males and females in 38 and 35 traits, respectively. On the other hand, analyses of geographic variability within each gender, i.e. analyses of variance and covariance of separate traits between localities showed “constant” dimensions and proportions of several body parts. Most of the invariable traits were the elements of the rear portion of the tortoises’ shell and free body parts, in both genders. Among these are some of the most prominent sexually dimorphic traits in T. hermanni. Therefore, we speculate that “standard” sizes of certain superficial bodily attributes in this species are conditioned by their role in gender discrimination, as well as courting and mating endeavours.

In terrestrial animals with rigid protective structures, the ability to upright after being overturned can make the difference between life and death, especially in suboptimal thermal conditions or in the presence of predators. This trait is assumed to be under strong selection. Different factors can influence righting ability, body dimensions and body mass for instance. As these morphological traits diverge among populations, inter-population variability in righting ability is expected. Previous studies on tortoises were performed within single populations and they usually focused on juveniles raised in captivity, precluding an assessment of the inter-population variability in a natural (realistic) context. In the current study, we quantified the righting performance in four populations of free-ranging adult tortoises. We found strong differences in righting success among populations and between genders, suggesting possible adaptations to local conditions. For instance, the topography (e.g. slopes) of each study site varied markedly. On average, males were more successful in righting themselves than females. Body size did not influence righting performances in males, but larger females were less successful compared to smaller ones. The success in righting was positively correlated with carapace domedness (height) and short bridges.

We processed 40 morphological characteristics of 676 adult Hermann's tortoises (Testudo hermanni) (310 males and 366 females) from four localities in the central parts of the Balkan Peninsula. Analyses of variance and covariance showed significant differences between males and females in 38 and 35 traits, respectively. On the other hand, analyses of geographic variability within each gender, i.e. analyses of variance and covariance of separate traits between localities showed "constant" dimensions and proportions of several body parts. Most of the invariable traits were the elements of the rear portion of the tortoises' shell and free body parts, in both genders. Among these are some of the most prominent sexually dimorphic traits in T hermanni. Therefore, we speculate that "standard" sizes of certain superficial bodily attributes in this species are conditioned by their role in gender discrimination, as well as courting and mating endeavours.

A population of dice snakes (Natrix tessellata) monitored since 2008 in a small island (18ha, 850m a.s.l., FYR of Macedonia) revealed unforeseen patterns for snakes living in temperate climates. More than 5000 individuals have been marked and the density is one of the highest ever recorded (>500 resident snakes per hectare). Reproductive and mortality rates are elevated, suggesting a high population turnover. These traits evoke a tropical rather than a temperate-climate ophidian demographic system. The population is highly polymorphic, three colour morphs (dotted, grey, and black) are observed in both sexes and each morph is represented by large numbers of individuals. This polymorphism pattern was not previously documented in snakes. Data obtained for other life history traits (e.g. body size, size at maturity, clutch size, diet, predation) markedly diverged in comparison to available information. Overall, our results reinforce the notion that the strong inter-population variability (often mediated by phenotypic plasticity) of snakes should be taken into account over large geographic scales; otherwise attempts to derive general patterns may well be strongly biased.

Background: Individuals of populations experiencing environmental and/or genetic disturbance during their development experience a reduction in buffering capacity of the developmental system. Such a reduction influences the level of developmental stability, which is often reflected in morphological traits such as asymmetry. Three kinds of asymmetry have been described: directional asymmetry (DA; i.e. left and right body side consistently differ from each other), fluctuating asymmetry (FA; i.e. subtle non-directional differences between left and right sides in bilaterally symmetrical traits), and antisymmetry (i.e. deviation from symmetry towards either the right or left sides). Fluctuating asymmetry is often used as an indicator of developmental stress. The variation of FA level is considered to be taxon, trait and stress specific. Accordingly, being exposed to various forms of environmental and/or genetic stress, the levels of FA may differ among demes. Moreover, different genetic constraints of two wing traits (lower heritability of wing size vs. higher heritability of wing shape) may influence the difference between island and mainland populations in the level of size FA would be more prominent than in the level of shape FA. Hypothesis: Island populations, which are often isolated and small, operate different evolutionary processes affecting developmental processes from those present in mainland populations. Hence, island populations experience significantly different FA compared with continental populations. Organism: The hoverfly, Merodon albifrons (Diptera, Syrphidae) Times and places: We collected a total of 60 female and 141 male specimens in spring 2009, 2010, and 2011 from the Greek islands of Lesbos (25 females and 38 males; May 2009), Chios (11 females and 60 males; May 2009), and Naxos (12 females and 13 males; May 2010). We also collected from the Greek mainland locality of Volos (12 females and 30 males; May 2009 and May 2011). Analytical methods: We used landmark-based geometric morphometrics to analyse level and patterns of asymmetry (FA, DA, and antisymmetry) in two wing traits (size and shape) within/ among populations. We completed morphometric and statistical analyses using the MorphoJ package (version 1.04a) (Klingenberg, 2011), R software 2.15.1 (R Development Core Team, 2008),

Hermann's tortoise (Testudo hermanni) is widely distributed in western and southern Europe. Most populations in the western part of the distribution range (e.g. Spain, France, Italy) are severely reduced, and relatively well studied, whilst the species is still abundant in eastern areas (i.e. the Balkans). However, essential biological information (e.g. main morphological, ecological, and behavioural characteristics) for the Balkans are still extremely limited. As reptiles exhibit strong geographic variation in most morphological, as well as life history traits, gathering data from distant areas is important. We present data from two populations of T. hermanni in Serbia, focusing on sexual dimorphism in body size and body shape. We found that almost all of the 43 morphological traits analysed were significantly different between sexes and that sexual size and sexual shape dimorphisms were not expressed in similar ways. Notably, sexual size dimorphism (SSD) was more pronounced than sexual shape dimorphism (SShD). Our analyses suggested that SShD is more stable than SSD, and that the scale of the focus (i.e. whole body proportions versus morphological details) is a key factor to test this notion. When general measurements were considered, the expected consistency of SShD between populations was verified; nevertheless, when more specific morphological attributes were considered, substantial variations were observed. These results provide a baseline for comparisons between populations to further examine geographic variation of sexual dimorphism.