Impact of mountain chains, sea straits and peripheral populations on genetic and taxonomic structure of a freshwater turtle, Mauremys leprosa (Reptilia, Testudines, Geoemydidae)

Mauremys leprosa, distributed in Iberia and North‐west Africa, contains two major clades of mtDNA haplotypes. Clade A occurs in Portugal, Spain and Morocco north of the Atlas Mountains. Clade B occurs south of the Atlas Mountains in Morocco and north of the Atlas Mountains in eastern Algeria and Tunisia. However, we recorded a single individual containing a clade B haplotype in Morocco from north of the Atlas Mountains. This could indicate gene flow between both clades. The phylogenetically most distinct clade A haplotypes are confined to Morocco, suggesting both clades originated in North Africa. Extensive diversity within clade A in south‐western Iberia argues for a glacial refuge located there. Other regions of the Iberian Peninsula, displaying distinctly lower haplotype diversities, were recolonized from within south‐western Iberia. Most populations in Portugal, Spain and northern Morocco contain the most common clade A haplotype, indicating dispersal from the south‐western Iberian refuge, gene flow across the Strait of Gibraltar, and reinvasion of Morocco by terrapins originating in south‐western Iberia. This hypothesis is consistent with demographic analyses, suggesting rapid clade A population increase while clade B is represented by stationary, fragmented populations. We recommend the eight, morphologically weakly diagnosable, subspecies of M. leprosa be reduced to two, reflecting major mtDNA clades: Mauremys l. leprosa (Iberian Peninsula and northern Morocco) and M. l. saharica (southern Morocco, eastern Algeria and Tunisia). Peripheral populations could play an important role in evolution of M. leprosa because we found endemic haplotypes in populations along the northern and southern range borders. Previous investigations in another western Palearctic freshwater turtle (Emys orbicularis) discovered similar differentiation of peripheral populations, and phylogeographies of Emys orbicularis and Mauremys rivulata underline the barrier status of mountain chains, in contrast to sea straits, suggesting common patterns for western Palearctic freshwater turtles.     

Rapid identification of lymnaeid snails and their infection with Fasciola gigantica in Thailand

Freshwater snails of the family Lymnaeidae are the intermediate hosts of the liver fluke Fasciola worldwide. While distinct species have been identified at the molecular level in other parts of the world such data have not been published for Thailand. In this study we collected Lymnaeidae from different localities across Thailand and analyzed their 16S rDNA sequences as a molecular signature for classification. In addition to the ubiquitous Radix rubiginosa, we have confirmed the presence of Austropeplea viridis and Radix swinhoei, for the latter of which the ribosomal rDNA sequences are reported for the first time, in North-Thailand. Based on the obtained 16S rDNA data three primer pairs were designed that allowed rapid identification of these snail species by PCR. To determine their infection status, PCR primers for F.gigantica cathepsin L were used in parallel with the snail 16S rDNA species-specific primers in multiplex PCR analyses. Western blot analysis of total snail protein with a monoclonal anti-F.gigantica cathepsin L antibody confirmed positive cathepsin L PCR results. The developed diagnostic PCR will be of use in risk assessment for transmission of fascioliasis in Thailand.     

Host-Specific Phenotypic Plasticity of the Turtle Barnacle Chelonibia testudinaria: A Widespread Generalist Rather than a Specialist

Turtle barnacles are common epibionts on marine organisms. Chelonibia testudinaria is specific on marine turtles whereas C. patula is a host generalist, but rarely found on turtles. It has been questioned why C. patula, being abundant on a variety of live substrata, is almost absent from turtles. We evaluated the genetic (mitochondrial COI, 16S and 12S rRNA, and amplified fragment length polymorphism (AFLP)) and morphological differentiation of C. testudinaia and C. patula from different hosts, to determine the mode of adaptation exhibited by Chelonibia species on different hosts. The two taxa demonstrate clear differences in shell morphology and length of 4–6^th cirri, but very similar in arthropodal characters. Moreover, we detected no genetic differentiation in mitochondrial DNA and AFLP analyses. Outlier detection infers insignificant selection across loci investigated. Based on combined morphological and molecular evidence, we proposed that C. testudinaria and C. patula are conspecific, and the two morphs with contrasting shell morphologies and cirral length found on different host are predominantly shaped by developmental plasticity in response to environmental setting on different hosts. Chelonibia testudinaria is, thus, a successful general epibiotic fouler and the phenotypic responses postulated can increase the fitness of the animals when they attach on hosts with contrasting life-styles.     

Coexistence of <Emphasis Type="Italic">Placobdella costata</Emphasis> (Fr. Müller, 1846) (Hirudinida: Glossiphoniidae) and mud turtle <Emphasis Type="Italic">Emys orbicularis</Emphasis>

Emys orbicularis is receding from Europe, mainly due to anthropogenic habitat changes. Its parasite, Placobdella costata, is widely distributed within both the former and the present distribution range of the host. Though closely associated with the mud turtle, it may have other hosts (birds, amphibians, reptiles). Its reproductive period coincides with that of its host’s migration to the breeding grounds, thus facilitating dispersal of the parasite. Based on literature data we have analyzed the geographic spread of P. costata and mud turtle to observe the possible overlap of their habitats. Observations on the population of mud turtle and the associated leech species (P. costata) were carried out in eastern Poland — Podlasie Lowland. The studies were conducted in spring and summer in 1986–1993. The leeches were collected from the turtles caught in the water and on land. Observations showed that most leeches were found on turtles inhabiting the lake or moving to a breeding area. The greatest intensity of invasion was observed in June and July and that most leeches were observed in female E. orbicularis characterized by greater length of the carapace and weight, compared with males and juvenile individuals.     

Distribution of mitochondrial haplotypes (cytb) in Polish populations of Emys orbicularis (L., 1758)

The European pond turtle, Emys orbicularis, inhabits a wide distribution area in the western Palaearctic. Polish populations of pond turtle represent the nominotypical subspecies Emys orbicularis orbicularis. The mitochondrial DNA haplotype (cytb gene) variation among 131 turtles from 26 locations in five regions of Poland was investigated. Five haplotypes belonging to three distinct lineages were identified. Two clades (I and II) were represented by two haplotypes each, while the other clade (IV) was represented by one haplotype. Three haplotypes were reported for the first time in E. orbicularis. The eastern part of Poland is inhabited exclusively by turtles bearing haplotype Ia. The remaining four sequence variants were recorded in western Poland where only the IIb haplotype is considered endemic. The distribution of the other haplotypes in western Poland could thus reflect past introductions or accidental releases. The authors regarded the two locations (Drzeczkowo and Karpicko) that were first included in the western Poland populations as autochthonous catchment areas of haplotype Ia.     

Native or not? Tracing the origin of wild-caught and captive freshwater turtles in a threatened and widely distributed species (Emys orbicularis)

Pet and food trade are among the major threats for many chelonians species worldwide. We investigate the impact of the present and past trade in European pond turtles (Emys orbicularis, E. trinacris) on their extant distribution pattern. Using a comprehensive mtDNA dataset of cytochrome b haplotypes derived from more than 1,550 individuals, we assigned wild-caught allochthonous and captive turtles from six European countries to their native regions across the entire distribution range. We found allochthonous haplotypes in all countries surveyed, providing evidence of long-distance translocations owing to past and present trade, illegal poaching or collection by tourists. In summary, we identify source regions of allochthonous turtles and past and present trade routes. Moreover, we point out future directions which would contribute to conservation and management of these threatened species.     

Haemogregarina pseudemydis n. sp. (Apicomplexa: Haemogregarinidae) and Pirhemocyton chelonarum n. sp. in Turtles from Louisiana*

SYNOPSIS. The blood of each of 95 turtles (8 species) collected from southeastern Louisiana was infected with some or all of the merogonic stages and gametocyte stage of Haemogregarina pseudemydis n. sp. Five species of turtles harbored Pirhemocyton chelonarum n. sp. Turtle Haemogregarina and Pirhemocyton are locality records for Louisiana. Pirhemocyton is reported for the first time in turtles and in the continental U.S.A.     

Where are you from,stranger? The enigmatic biogeography of North African pond turtles (Emys orbicularis)

The European pond turtle (Emys orbicularis) is a Nearctic element in the African fauna and thought to have invaded North Africa from the Iberian Peninsula. All North African populations are currently identified with the subspecies E. o. occidentalis. However, a nearly range-wide sampling in North Africa used for analyses of mitochondrial and microsatellite DNA provides evidence that only Moroccan populations belong to this taxon, while eastern Algerian and Tunisian pond turtles represent an undescribed distinct subspecies. These two taxa are most closely related to E. o. galloitalica with a native distribution along the Mediterranean coast of northern Spain through southern France to western and southern Italy. This group is sister to a clade comprising several mitochondrial lineages and subspecies of E. orbicularis from Central and Eastern Europe plus Asia, and the successive sisters are E. o. hellenica and E. trinacris. Our results suggest that E. orbicularis has been present in North Africa longer than on the Iberian Peninsula and that after an initial invasion of North Africa by pond turtles from an unknown European source region, there was a phase of diversification in North Africa, followed by a later re-invasion of Europe by one of the African lineages. The differentiation of pond turtles in North Africa parallels a general phylogeographic paradigm in amphibians and reptiles, with deeply divergent lineages in the western and eastern Maghreb. Acknowledging their genetic similarity, we propose to synonymize the previously recognized Iberian subspecies E. o. fritzjuergenobsti with E. o. occidentalis sensu stricto. The seriously imperiled Moroccan populations of E. o. occidentalis represent two Management Units different in mitochondrial haplotypes and microsatellite markers. The conservation status of eastern Algerian pond turtles is unclear, while Tunisian populations are endangered. Considering that Algerian and Tunisian pond turtles represent an endemic taxon, their situation throughout the historical range should be surveyed to establish a basis for conservation measures.     

Genomic Diversity within the Genus Pediococcus as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Ultrastructure of the cyst wall of Sarcocystis spp. from some rodents in Malaysia.

The cyst wall of Sarcocystis cysts from the skeletal muscles and subcutaneous tissues of 4 species of rats and 1 species of bandicoot in Malaysia was examined under the electron microscope. Three types of sarcosysts with morphologically distinct cyst walls were found in these rodents. These morphologically distinct types of sarcocysts occurred as single or mixed infections in their various rodent hosts, suggesting that these rodents are important, though non-specific, intermediate hosts in the life cycle of at least 3 species of Sarcocystis. The final hosts of these Sarcocystis species are unknown.     

A new cryptic species of pond turtle from southern Italy, the hottest spot in the range of the genus Emys (Reptilia, Testudines, Emydidae)

Geographic variation in the mtDNA haplotypes (cytochrome b gene) of 127 European pond turtles from Italy was investigated. Thirty‐eight of the Italian samples were also studied by nuclear fingerprinting (ISSR PCR) and compared with samples from other parts of the range representing all nine currently known mtDNA lineages of Emys orbicularis. Our genetic findings were compared against morphological data sets (measurements, colour pattern) for 109 adult turtles from southern Italy. Italy is displaying on a small geographical scale the most complicated variation known over the entire distributional area of Emys (North Africa over Europe and Asia Minor to the Caspian and Aral Seas). The Tyrrhenic coast of the Apennine Peninsula, the Mt. Pollino area and Basilicata are inhabited by Emys orbicularis galloitalica, a subspecies harbouring a distinct mtDNA lineage. The same lineage is also found in Sardinia. Along the Adriatic coast of Italy and on the Salentine Peninsula (Apulia, southern Italy), another morphologically distinctive subspecies (Emys orbicularis hellenica) occurs, which also bears a different mtDNA lineage. A higher diversity of mtDNA haplotypes in the south of the Apennine Peninsula suggests that the glacial refugia of E. o. galloitalica and E. o. hellenica were located here. A further refuge of E. o. hellenica probably existed in the southern Balkans. The west coasts of the Balkans and Corfu have probably been colonized from Italy and not from the geographically closer southern Balkanic refuge. In Sicily, a third mtDNA lineage is distributed, which is sister to all other known lineages of Emys. Morphologically, Sicilian pond turtles resemble E. o. galloitalica. However, nuclear fingerprinting revealed a clear distinctiveness of the Sicilian taxon, whereas no significant divergence was detected between representatives of the other eight mtDNA lineages of Emys. Furthermore, nuclear fingerprinting provided no evidence for current or past gene flow between the Sicilian taxon and the mainland subspecies of E. orbicularis. Therefore, Sicilian pond turtles are described here as a species new to science. Some populations in Calabria and on the Salentine Peninsula comprise individuals of different mtDNA lineages. We interpret this as a natural contact. However, we cannot exclude that these syntopic occurrences are the result of human activity. For example, in other parts of Italy, the natural distribution pattern of Emys is obscured by allochthonous turtles. This could also be true for southern Italy. The discovery of the complex taxonomic differentiation in southern Italy requires reconsidering conservation strategies.     

Basic morphological data of native Czech Emys orbicularis revealed by subfossil finds

Holocene Czech Emys orbicularis are rare in museum collections because the species disappeared from this region centuries ago. Fifteen reconstructed shells of subfossil Czech specimens of E. orbicularis (ranging in age from 3800–3500 BC to 1300 AD) from archaeological sites were used to assess basic morphological data. Our results provide evidence that the extinct Czech turtles fall into the size range of extant Central European E. orbicularis populations from the Hungarian Lowland. Maximum male and female straight-line carapacial lengths of the Czech specimens reached at least 160 and 175 mm, respectively.     

Big data can cause big mistakes: using the Societas Europaea Herpetologica atlas by Sillero et al. (2014), the distribution of <Emphasis Type="Italic">Emys orbicularis</Emphasis> will be misunderstood

Problems with the reliability of the distribution map for Emys orbicularis in the online atlas of European amphibians and reptiles (NA2RE) are highlighted. Two distinct species (E. orbicularis, E. trinacris) are lumped together there. For E. orbicularis, many regions are included into the native distribution range where the species does not occur naturally. On the contrary, records are missing for vast parts of the native range. Using data from the NA2RE atlas cannot be recommended presently, especially not for species distribution modelling.