Pavel Široký

Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic differentiation in western Palaearctic tortoises (Testudo graeca complex; Testudines, Testudinidae)

Tortoises of the Testudo graeca complex inhabit a patchy range that covers part of three continents (Africa, Europe, Asia). It extends approximately 6500 km in an east-west direction from eastern Iran to the Moroccan Atlantic coast and about 1600 km in a north-south direction from the Danube Delta to the Libyan Cyrenaica Peninsula. Recent years have seen a rapid increase of recognized taxa. Based on morphological investigations, it was suggested that this group consists of as many as 20 distinct species and is paraphyletic with respect to T. kleinmanni sensu lato and T. marginata . Based on samples from representative localities of the entire range, we sequenced the mitochondrial cytochrome b gene and conducted nuclear genomic fingerprinting with ISSR PCR. The T. graeca complex is monophyletic and sister to a taxon consisting of T. kleinmanni sensu lato and T. marginata . The T. graeca complex comprises six well-supported mtDNA clades (A-F). Highest diversity is found in the Caucasian Region, where four clades occur in close neighbourhood. This suggests, in agreement with the fossil record, the Caucasian Region as a radiation centre. Clade A corresponds to haplotypes from the East Caucasus. It is the sister group of another clade (B) from North Africa and western Mediterranean islands. Clade C includes haplotypes from western Asia Minor, the southeastern Balkans and the western and central Caucasus Region. Its sister group is a fourth, widely distributed clade (D) from southern and eastern Asia Minor and the Levantine Region (Near East). Two further clades are distributed in Iran (E, northwestern and central Iran; F, eastern Iran). Distinctness of these six clades and sister group relationships of (A + B) and (C + D) are well-supported; however, the phylogeny of the resulting four clades (A + B), (C + D), E and F is poorly resolved. While in a previous study (Fritz et al., 2005a) all traditionally recognized Testudo species were highly distinct using mtDNA sequences and ISSR fingerprints, we detected within the T. graeca complex no nuclear genomic differentiation paralleling mtDNA clades. We conclude that all studied populations of the T. graeca complex are conspecific under the Biological Species Concept. There is major incongruence between mtDNA clades and morphologically defined taxa. Morphologically well-defined taxa, like T. g. armeniaca or T. g. floweri , nest within clades comprising also geographically neighbouring, but morphologically distinctive populations of other taxa (clade A: T. g. armeniaca , T. g. ibera , T. g. pallasi ; clade D: T. g. anamurensis , T. g. antakyensis , T. g. floweri , T. g. ibera , T. g. terrestris ), while sequences of morphologically similar tortoises of the same subspecies ( T. g. ibera sensu stricto or T. g. ibera sensu lato) scatter over two or three genetically distinct clades (A, C or A, C, D, respectively). This implies that pronounced morphological plasticity, resulting in phenotypes shaped by environmental pressure, masks genetic differentiation. To achieve a more realistic taxonomic arrangement reflecting mtDNA clades, we propose reducing the number of T. graeca subspecies considerably and regard in the eastern part of the range five subspecies as valid ( T. g. armeniaca , T. g. buxtoni , T. g. ibera , T. g. terrestris , T. g. zarudnyi ). As not all North African taxa were included in the present study, we refrain from synonymizing North African taxa with T. g. graeca (mtDNA clade B) that represents a further valid subspecies.

Mitochondrial phylogeography of European pond turtles ( Emys orbicularis , Emys trinacris ) – an update

Based on more than 1100 samples of Emys orbicularis and E. trinacris, data on mtDNA diversity and distribution of haplotypes are provided, including for the first time data for Armenia, Georgia, Iran, and the Volga, Ural and Turgay River Basins of Russia and Kazakhstan. Eight mitochondrial lineages comprising 51 individual haplotypes occur in E. orbicularis, a ninth lineage with five haplotypes corresponds to E. trinacris. A high diversity of distinct mtDNA lineages and haplotypes occurs in the south, in the regions where putative glacial refuges were located. More northerly parts of Europe and adjacent Asia, which were recolonized by E. orbicularis in the Holocene, display distinctly less variation; most refuges did not contribute to northern recolonizations. Also in certain southern European lineages a decrease of haplotype diversity is observed with increasing latitude, suggestive of Holocene range expansions on a smaller scale.

A rangewide phylogeography of Hermann's tortoise, Testudo hermanni (Reptilia: Testudines: Testudinidae): implications for taxonomy

Hermanns tortoise (Testudo hermanni), the best‐known western Palaearctic tortoise species, has a rare natural distribution pattern comprising the Mediterranean areas of the Iberian, Apennine, and Balkan Peninsulas, as well as Sicily, Corsica and Sardinia. The western part of this range is traditionally considered habitat for T. h. hermanni, while T. h. boettgeri occurs in the Balkans. Taxonomy of this tortoise has been challenged in recent years, with the two subspecies being considered full species and the central Dalmatian populations of T. h. boettgeri being considered a third species, T. hercegovinensis. Using an mtDNA fragment approximately 1150 bp long (cytochrome b gene and adjacent portion of tRNA‐Thr gene), we investigated mtDNA diversity with regard to contrasting concepts of two subspecies or three species. Seven closely related haplotypes were identified from the western Mediterranean and 15 different, in part much‐differentiated, haplotypes from the Balkans. Western Mediterranean haplotypes differ from Balkan haplotypes in 16–42 mutation steps. One to seven mutation steps occur within western Mediterranean populations. Balkan haplotypes, differing in 1−37 nucleotides, group in parsimony network analysis into three major assemblages that display, in part, a similar degree of differentiation to that of western Mediterranean haplotypes relative to Balkan haplotypes. Rates of sequence evolution are different in both regions, and low divergence, palaeogeography and the fossil record suggest a slower molecular clock in the western Mediterranean. While monophyly in western Mediterranean haplotypes is well‐supported, conflicting evidence is obtained for Balkan haplotypes; maximum parsimony supports monophyly of Balkan haplotypes, but other phylogenetic analyses (Bayesian, ML, ME) indicate Balkan haplotypes could be paraphyletic with respect to the western Mediterranean clade. These results imply a process of differentiation not yet complete despite allopatry in the western Mediterranean and the Balkans, and suggest all populations of T. hermanni are conspecific. In the western Mediterranean no clear geographical pattern in haplotype distribution is found. Distribution of Balkan haplotypes is more structured. One group of similar haplotypes occurs in the eastern Balkans (Bulgaria, Republic of Macedonia, Romania and the Greek regions Evvia, Macedonia, Peloponnese, Thessaly and Thrace). Two distinct haplotypes, differing in eight to nine mutation steps from the most common haplotype of the first group, are confined to the western slope of the Taygetos Mts. in the Peloponnese. Yet another group, connected over between four and 23 mutation steps with haplotypes of the eastern Balkan group, occurs along the western slope of the Dinarid and Pindos Mts. (Istria, Dalmatia, western Greece). Taygetos haplotypes are nested within other haplotypes in all phylogenetic analyses and support for monophyly of the other Balkan groups is at best weak. We conclude that using the traditional two subspecies model should be continued for T. hermanni. Phylogeographies of T. hermanni and Emys orbicularis, another codistributed chelonian, are markedly different, but share a few similarities. Both were forced to retreat to southern refuges during Pleistocene glaciations. With the advent of Holocene warming, E. orbicularis underwent rapid range expansion and temperate regions of Europe and adjacent Asia were recolonized from refuges in the Balkans and the northern Black Sea Region. By contrast, T. hermanni remained more or less confined to refuges and nearby regions, resulting in a much smaller range, and allopatric and parapatric distribution of haplotype groups and clades. MtDNA lineages are more diverse in E. orbicularis than they are in T. hermanni on southern European peninsulas, indicating several distinct glacial refuges in close proximity and extensive intergradation during Holocene range expansion for E. orbicularis. In T. hermanni it is likely that only on the Balkan Peninsula was more than one refuge located, corresponding to the parapatric ranges of haplotype groups currently there. On the old western Mediterranean islands Corsica and Sardinia no differentiated (E. orbicularis) or only weakly differentiated haplotypes (T. hermanni) occur, even though there is evidence for the presence of both species on Corsica since at least the Middle Pleistocene. High mountain chains constitute major barriers separating distinct mtDNA clades or groups in each species.

Mitochondrial phylogeography of Testudo graeca in the Western Mediterranean: Old complex divergence in North Africa and recent arrival in Europe

We investigated the mitochondrial phylogeography of spur-thighed tortoises (Testudo graeca) in the Western Mediterranean. In North Africa, four major lineages (A-D) occur that together constitute a well-supported clade corresponding to one of the six major clades within T. graeca; the North African clade is sister to a Caucasian clade representing the subspecies T. g. armeniaca. Phylogenetic relationships between the North African lineages are badly resolved. Lineage A is distributed in Tunisia and adjacent Algeria, lineage B in Algeria and northern Morocco, lineage C in the Libyan Cyrenaica Peninsula, and lineage D north of the High Atlas Mts. and in the Souss Valley (southern Morocco). Lineage B is subdivided into two subgroups, B1 (eastern Morocco and Algeria) and B2 (north-western Morocco). Italian tortoises harbour haplotypes of lineage A, Spanish tortoises of subgroup B1. Based on a relaxed molecular clock calibrated with fossil evidence, the six major mtDNA clades of T. graeca are estimated to have diverged approximately 4.2-1.8 Ma ago; the split between the clades representing the eastern subspecies T. g. ibera and T. g. terrestris is younger than the split between Western Mediterranean tortoises and T. g. armeniaca. The Western Mediterranean lineages A-D were dated to have diverged at least 1.4-1.1 Ma ago; B1 and B2 split approximately 0.7 Ma ago. Our results suggest that Italian and Spanish tortoises were either introduced or originated from trans-oceanic dispersal in historic or prehistoric times. Spur-thighed tortoises invaded North Africa probably across Near Eastern landbridges that emerged in the Late Tertiary. Their diversification in North Africa seems to be correlated with habitat aridization cycles during the Pleistocene. The ranges of the Western Mediterranean lineages largely correspond to the distribution of morphologically defined subspecies in North Africa, with exception of T. g. graeca and T. g. whitei, and of T. g. lamberti and T. g. marokkensis, which are not differentiated. We propose to lump the first two subspecies under the name of T. g. graeca and the latter under the name of T. g. marokkensis. The complex differentiation of spur-thighed tortoises in North Africa implies that the model of a bipartite east-west differentiation, as proposed for other Maghrebian amphibians and reptiles, may be too simplistic, reflecting incomplete locality sampling rather than actual phylogeographic differentiation.

Unexpected early extinction of the European pond turtle (Emys orbicularis) in Sweden and climatic impact on its Holocene range

Using ancient DNA sequences of subfossil European pond turtles (Emys orbicularis) from Britain, Central and North Europe and accelerator mass spectrometry radiocarbon dating for turtle remains from most Swedish sites, we provide evidence for a Holocene range expansion of the pond turtle from the southeastern Balkans into Britain, Central Europe and Scandinavia, according to the ‘grasshopper pattern’ of Hewitt. Northeastern Europe and adjacent Asia were colonized from another refuge located further east. With increasing annual mean temperatures, pond turtles reached southern Sweden approximately 9800 years ago. Until approximately 5500 years ago, rising temperatures facilitated a further range expansion up to Östergötland, Sweden (approximately 58°30′N). However, around 5500 years ago pond turtle records suddenly terminate in Sweden, some 1500 years before the Holocene thermal maximum ended in Scandinavia and distinctly earlier than previously thought. This extinction coincides with a temporary cooling oscillation during the Holocene thermal maximum and is likely related to lower summer temperatures deteriorating reproductive success. Although climatic conditions improved later again, recolonization of Sweden from southern source populations was prevented by the Holocene submergence of the previous land connection via the Danish Straits that occurred approximately 8500 years ago.

Hyalomma aegyptium as dominant tick in tortoises of the genus Testudo in Balkan countries, with notes on its host preferences

Collection of 1327 ticks sampled throughout Greece, Bulgaria, Romania and Croatia, from 211 tortoises belonging to three species, Testudo marginata Schoepff, T. graeca Linnaeus, and T. hermanni Gmelin, revealed the presence of four species of ixodid ticks, namely Hyalomma aegyptium (Linnaeus), Haemaphysalis sulcata Canestrini and Fanzago, H. inermis Birula and Rhipicephalus sanguineus (Latreille). Study confirmed the strong dominance of all life stages of H. aegyptium among ticks parasitizing west Palaearctic tortoises of genus Testudo Linnaeus. Furthermore, a considerable portion of ticks collected from tortoises in southwestern Bulgaria represent larvae and nymphs of H. sulcata. At the same area we collected as exception one larva and one nymph of H. inermis from a single specimen of T. hermanni. Our findings of four adults of R. sanguineus is the first record of this species from reptilian host. According to our results achieved on localities with syntopic occurrence of two tortoise species, T. marginata and T. graeca represent in the Balkans the principal hosts of H. aegyptium, whereas T. hermanni serves only as an alternative host in the areas close to range of either T. marginata or T. graeca.

The distribution and spreading pattern of Dermacentor reticulatus over its threshold area in the Czech Republic—How much is range of this vector expanding?

Host-seeking Dermacentor reticulatus ticks were detected by flagging method at 46 localities at south-east part of the Czech Republic, in the basins of rivers Morava and Dyje. Exact north-west distribution limits of D. reticulatus were defined in this area for the first time. Detailed prediction map of probabilities of D. reticulatus occurrence was obtained using GIS analysis. Spatial model delimited a south-north gradient in probability across the studied area, with highest probabilities above 0.8 in its southernmost part. Abundance of D. reticulatus varied markedly between localities in interval 0.33-222 of ticks per flag per hour. The highest abundances were in flooded areas at lower streams, towards upper streams abundance and density of these ticks decreased. Females prevailed in samples with population sex ratio of 0.413, significantly deviating from parity. Larvae and nymphs of this species were not detected by flagging. Although D. reticulatus range expansion probably did not reach such a degree as reported in other countries, these ticks became very abundant in some parts of studied area. Since spreading of vector-borne diseases became a problem in Europe, the knowledge of their exact recent geographic ranges is important for future modelling of their shift predictability.

Dermacentor reticulatus: a vector on the rise

Dermacentor reticulatus is a hard tick species with extraordinary biological features. It has a high reproduction rate, a rapid developmental cycle, and is also able to overcome years of unfavourable conditions. Dermacentor reticulatus can survive under water for several months and is cold-hardy even compared to other tick species. It has a wide host range: over 60 different wild and domesticated hosts are known for the three active developmental stages. Its high adaptiveness gives an edge to this tick species as shown by new data on the emergence and establishment of D. reticulatus populations throughout Europe. The tick has been the research focus of a growing number of scientists, physicians and veterinarians. Within the Web of Science database, more than a fifth of the over 700 items published on this species between 1897 and 2015 appeared in the last three years (2013–2015). Here we attempt to synthesize current knowledge on the systematics, ecology, geographical distribution and recent spread of the species and to highlight the great spectrum of possible veterinary and public health threats it poses. Canine babesiosis caused by Babesia canis is a severe leading canine vector-borne disease in many endemic areas. Although less frequently than Ixodes ricinus, D. reticulatus adults bite humans and transmit several Rickettsia spp., Omsk haemorrhagic fever virus or Tick-borne encephalitis virus. We have not solely collected and reviewed the latest and fundamental scientific papers available in primary databases but also widened our scope to books, theses, conference papers and specialists colleagues’ experience where needed. Besides the dominant literature available in English, we also tried to access scientific literature in German, Russian and eastern European languages as well. We hope to inspire future research projects that are necessary to understand the basic life-cycle and ecology of this vector in order to understand and prevent disease threats. We conclude that although great strides have been made in our knowledge of the eco-epidemiology of this species, several gaps still need to be filled with basic research, targeting possible reservoir and vector roles and the key factors resulting in the observed geographical spread of D. reticulatus.

Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. megalocephala)

Grass snakes (Natrix natrix) represent one of the most widely distributed snake species of the Palaearctic region, ranging from the North African Maghreb region and the Iberian Peninsula through most of Europe and western Asia eastward to the region of Lake Baikal in Central Asia. Within N. natrix, up to 14 distinct subspecies are regarded as valid. In addition, some authors recognize big‐headed grass snakes from western Transcaucasia as a distinct species, N. megalocephala. Based on phylogenetic analyses of a 1984‐bp‐long alignment of mtDNA sequences (ND4+tRNAs, cyt b) of 410 grass snakes, a nearly range‐wide phylogeography is presented for both species. Within N. natrix, 16 terminal mitochondrial clades were identified, most of which conflict with morphologically defined subspecies. These 16 clades correspond to three more inclusive clades from (i) the Iberian Peninsula plus North Africa, (ii) East Europe and Asia and (iii) West Europe including Corso‐Sardinia, the Apennine Peninsula and Sicily. Hypotheses regarding glacial refugia and postglacial range expansions are presented. Refugia were most likely located in each of the southern European peninsulas, Corso‐Sardinia, North Africa, Anatolia and the neighbouring Near and Middle East, where the greatest extant genetic diversity occurs. Multiple distinct microrefugia are inferred for continental Italy plus Sicily, the Balkan Peninsula, Anatolia and the Near and Middle East. Holocene range expansions led to the colonization of more northerly regions and the formation of secondary contact zones. Western Europe was invaded from a refuge within southern France, while Central Europe was reached by two distinct range expansions from the Balkan Peninsula. In Central Europe, there are two contact zones of three distinct mitochondrial clades, and one of these contact zones was theretofore completely unknown. Another contact zone is hypothesized for Eastern Europe, which was colonized, like north‐western Asia, from the Caucasus region. Further contact zones were identified for southern Italy, the Balkans and Transcaucasia. In agreement with previous studies using morphological characters and allozymes, there is no evidence for the distinctiveness of N. megalocephala. Therefore, N. megalocephala is synonymized with N. natrix.

West-to-east differences of Babesia canis canis prevalence in Dermacentor reticulatus ticks in Slovakia

Babesia canis canis is the most frequent causative agent of canine babesiosis in Central Europe, frequently causing severe disease. Recently, many new endemic foci of this disease have been reported from European countries. Growing incidence of canine babesiosis was recorded also in Slovakia during the last decade, from first cases in eastern Slovakia ten years ago to recent cases all over the south of the country. We have used nested PCR-RFLP method to study prevalence of B. c. canis in its natural tick vector Dermacentor reticulatus, collected at three geographically isolated lowland areas of southern Slovakia situated in the southeast, southwest, and west of Slovakia, respectively. The highest prevalence of B. c. canis was observed in D. reticulatus from eastern Slovakia (14.7%; n=327), whereas the prevalence in southwest was significantly lower (2.3%; n=1205). Notably, all 874 D. reticulatus ticks collected at Záhorská nížina lowland (W Slovakia) were B. c. canis-negative. Recorded differences in Babesia prevalence concurs well with the shift in incidence of clinical cases of canine babesiosis as observed by vet practitioners. Presented results revealed that eastern Slovakia represents an area of high risk of B. c. canis infection, whereas western areas of the country still remain Babesia canis-free.