Mediterranean vineyards and olive groves in Croatia harbour some rare and endemic invertebrates.

The Mediterranean is characterised by high biodiversity and numerous endemic species. These species are not only present in natural habitats, but also inhabit areas under human influence, such as agricultural lands. In the biodiversity assessment of Mediterranean vineyards and olive orchards within Zadar County, in Croatia, we identified eight endemic species with Mediterranean distribution, six with a Balkan Peninsula distribution, four with Dinaric Alps distribution and three species rare and endangered in Europe. Alongside these species, we have recorded five new species for Croatian fauna, many of those identified by combining morphological characteristics and the DNA barcoding tool. Araneae and Coleoptera contributed the highest number of endemic species and groups with new record were the following: Coleoptera, Diptera and Araneae. Compared to other sites, an olive orchard with ecological pest management (EPM), surrounded by natural ecosystems, had the highest ratio of endemic and rare species. Our findings emphasise that agricultural lands in the Mediterranean can be habitats for endemic and rare species and that future biodiversity research of these habitats is highly important, to monitor potential biodiversity changes and motivate future species and ecosystem conservation.

A new set of microsatellite markers for Phoxinus lumaireul senso lato, Phoxinus marsilii and Phoxinus krkae for population and molecular taxonomic studies.

Minnows of the genus Phoxinus are common and an often highly abundant fish species in Palearctic freshwater habitats. Phoxinus species have a complex evolutionary history, phylogenetic relationships are not well understood and there are a number of unresolved taxonomic problems. There are currently 23 different mitochondrial genetic lineages identified in the genus Phoxinus, 13 of which are recognized as valid species. The taxonomic status of these lineages requires resolution, including the degree to which they can interbreed. Suitable nuclear molecular markers for studies of population divergence and interbreeding between morphotypes and mitochondrial lineages are lacking for Phoxinus species. Therefore, the authors developed a set of microsatellite markers using genomic information from Phoxinus lumaireul and tested their suitability for this and two related species, Phoxinus krkae and Phoxinus marsilii. Out of 16 microsatellite candidate loci isolated, 12 were found to be in Hardy-Weinberg equilibrium when tested on two P. lumaireul senso lato populations. Seven loci amplified across the three species, enabling the study of intraspecific genetic diversity and population structure within P. marsilii and P. krkae. The markers were able to clearly resolve differences among the three tested species, including the recently described P. krkae, and are therefore suitable for the detection of introgression and hybridization among populations consisting of mixtures of two or more of P. lumaireul s. l., P. marsilii and P. krkae.

Iguanainsularis (Iguanidae) from the southern Lesser Antilles: An endemic lineage endangered by hybridization.

The newly described horned iguanaIguanainsularis from the southern Lesser Antilles is separated in two easily recognized subspecies: I.insularissanctaluciae from St. Lucia and I.insularisinsularis from the Grenadines. Its former description is completed by the use of 38 new samples for genetic and morphological analysis. Seventeen microsatellites were used to estimate genetic diversity, population structure and the level of introgression with other Iguana species over nearly the whole range of the species. ND4 and PAC sequences were also used to better characterize hybridization and to complete the description of this lineage. The I.insularis population of St. Vincent shows a high level of introgression from I.iguana whereas in the Grenadines, most islands present pure insularis populations but several show evidence of introgressions. Of the two remaining populations of I.insularissanctaluciae, only one is still purebred. The recent identification of this and other distinct insular species and subspecies in the eastern Caribbean, and evaluation of where hybridization has occurred, are timely and important because the native iguanas are in urgent need of conservation action. Among the greatest threats is the ongoing human-mediated spread of invasive iguanas from Central and South America, which are destroying the endemic insular lineages through multiple diachronic introgression events.

Genetic diversity and structure of the noble crayfish populations in the Balkan Peninsula revealed by mitochondrial and microsatellite DNA markers.

BACKGROUND: The noble crayfish (Astacus astacus) is a native European species in decline, with a contracting range and diminishing populations and abundance. Previous studies revealed this species significant genetic diversity in the south-eastern Europe, with populations from the western and the southern part of the Balkan Peninsula being the most divergent. However, sampling of populations from the western part of the Balkans was limited and insufficient for investigating genetic diversity and population divergence for the purpose of conservation planning and management. Thus, the major aim of this study was to fill in this knowledge gap by studying mitochondrial and microsatellite DNA diversity, using 413 noble crayfish from 18 populations from waterbodies in the western part of the Balkan Peninsula. METHODS: Phylogenetic analysis of studied populations and their mitochondrial diversity were studied using COI and 16S sequences and population genetic structure was described using 15 microsatellite loci. RESULTS: Phylogeographic analysis revealed new divergent mitochondrial haplotypes for the populations in the westernmost part of the Balkan Peninsula in the tributaries of the Sava and Drava rivers. Microsatellite data indicated that these populations harbour an important component of genetic diversity within A. astacus. The results suggest that the western part of the Balkans played an important role as microrefugia during the Pleistocene climate fluctuations, allowing the long term persistence of A. astacus populations in this region. These results will also be important to supporting conservation decision making and planning.

New insights into the genetic diversity of the stone crayfish: taxonomic and conservation implications.

BACKGROUND: Austropotamobius torrentium is a freshwater crayfish species native to central and south-eastern Europe, with an intricate evolutionary history and the highest genetic diversity recorded in the northern-central Dinarides (NCD). Its populations are facing declines, both in number and size across its entire range. By extanding current knowledge on the genetic diversity of this species, we aim to assist conservation programmes. Multigene phylogenetic analyses were performed using different divergence time estimates based on mitochondrial and, for the first time, nuclear DNA markers on the largest data set analysed so far. In order to reassess taxonomic relationships within this species we applied several species delimitation methods and studied the meristic characters with the intention of finding features that would clearly separate stone crayfish belonging to different phylogroups. RESULTS: Our results confirmed the existence of high genetic diversity within A. torrentium, maintained in divergent phylogroups which have their own evolutionary dynamics. A new phylogroup in the Kordun region belonging to NCD has also been discovered. Due to the incongruence between implemented species delimitation approaches and the lack of any morphological characters conserved within lineages, we are of the opinion that phylogroups recovered on mitochondrial and nuclear DNA are cryptic subspecies and distinct evolutionary significant units. CONCLUSIONS: Geographically and genetically isolated phylogroups represent the evolutionary legacy of A. torrentium and are highly relevant for conservation due to their evolutionary distinctiveness and restricted distribution.

Continental-scale patterns of hyper-cryptic diversity within the freshwater model taxon Gammarus fossarum (Crustacea, Amphipoda).

Traditional morphological diagnoses of taxonomic status remain widely used while an increasing number of studies show that one morphospecies might hide cryptic diversity, i.e. lineages with unexpectedly high molecular divergence. This hidden diversity can reach even tens of lineages, i.e. hyper cryptic diversity. Even well-studied model-organisms may exhibit overlooked cryptic diversity. Such is the case of the freshwater crustacean amphipod model taxon Gammarus fossarum. It is extensively used in both applied and basic types of research, including biodiversity assessments, ecotoxicology and evolutionary ecology. Based on COI barcodes of 4926 individuals from 498 sampling sites in 19 European countries, the present paper shows (1) hyper cryptic diversity, ranging from 84 to 152 Molecular Operational Taxonomic Units, (2) ancient diversification starting already 26 Mya in the Oligocene, and (3) high level of lineage syntopy. Even if hyper cryptic diversity was already documented in G. fossarum, the present study increases its extent fourfold, providing a first continental-scale insight into its geographical distribution and establishes several diversification hotspots, notably south-eastern and central Europe. The challenges of recording hyper cryptic diversity in the future are also discussed.

Successful post-glacial colonization of Europe by single lineage of freshwater amphipod from its Pannonian Plio-Pleistocene diversification hotspot.

Gammarus roeselii Gervais, 1835 is a morphospecies with a wide distribution range in Europe. The Balkan Peninsula is known as an area of pre-Pleistocene cryptic diversification within this taxon, resulting in at least 13 Molecular Operational Taxonomic Units (MOTUs). The morphospecies diversified there during Neogene and has probably invaded other parts of the continent very recently, in postglacial or even historical times. Thus, the detailed goals of our study were to (1) identify which lineage(s) colonized Central-Western Europe (CWE), (2) determine their possible geographical origin, (3) verify, whether the colonisation was associated with demographic changes. In total, 663 individuals were sequenced for the cytochrome oxidase I (COI) barcoding fragment and 137 individuals for the internal transcribed spacer II (ITS2). We identified two MOTUs in the study area with contrasting Barcode Index Number and haplotype diversities. The Pannonian Basin (PB) appeared to be a potential ice age refugium for the species, while CWE was colonised by a single lineage (also present in PB), displaying low genetic diversity. Our results suggest that G. roeselii is a relatively recent coloniser in CWE, starting demographic expansion around 10 kya.

Painted black: Iguana melanoderma (Reptilia, Squamata, Iguanidae) a new melanistic endemic species from Saba and Montserrat islands (Lesser Antilles).

The Lesser Antilles, in the Eastern Caribbean, is inhabited by three Iguana species: the Lesser Antillean iguanaIguana delicatissima, which is endemic to the northernmost islands of the Lesser Antilles, the introduced common iguana from South America, Iguana iguana iguana, represented also by the two newly described endemic subspecies Iguana iguana sanctaluciae from Saint Lucia and Iguana iguana insularis from Saint Vincent and the Grenadines, and Grenada, and the introduced Iguana rhinolopha from Central America. Drawing on both morphological and genetic data, this paper describes the Iguana populations from Saba and Montserrat as a new species, Iguana melanoderma. This species is recognized on the basis of the following combination of characteristics: private microsatellite alleles, unique mitochondrial ND4 haplotypes, a distinctive black spot between the eye and tympanum, a dorsal carpet pattern on juveniles and young adults, a darkening of body coloration with aging (except for the anterior part of the snout), a black dewlap, pink on the jowl, the high number of large tubercular nape scales, fewer than ten medium sized-triangular dewlap spikes, high dorsal spikes, and lack of horns on the snout. This new melanistic taxon is threatened by unsustainable harvesting (including for the pet trade) and both competition and hybridization from escaped or released invasive alien iguanas (I. iguana iguana and I. rhinolopha) from South and Central America, respectively. The authors call for action to conserve Iguana melanoderma in Saba and Montserrat and for further research to investigate its relationship to other melanistic iguanas from the Virgin Islands and coastal islands of Venezuela.

Description of a new species of Phoxinus from the upper Krka River (Adriatic Basin) in Croatia (Actinopterygii: Leuciscidae), first discovered as a molecular clade.

Using morphometric, meristic and qualitative characters, we confirmed a specific status of a clade discovered based on molecular data only. The newly described species Phoxinus krkae has a very local distribution in the upper Krka River but is easily morphologically distinguished from geographically neighbouring species with much wider ranges: Phoxinus lumaireul, Phoxinus marsilii and Phoxinus csikii. Phoxinus krkae is well differentiated in statistical analyses and differs from other species of Phoxinus in northern Adriatic Sea drainages and right-bank tributaries of the middle and lower River Danube by a combination of characters, none of them unique: caudal peduncle length 1.9-2.2 times minimum depth of caudal peduncle; upper lip not projecting beyond lower lip; rostral fold absent; total number of scales in lateral series 74-84; lateral line incomplete and interrupted, commonly 17-49 (< 60% of total number of scales in lateral series) with no or few canal segments on posterior body and caudal peduncle; scales on belly extending forward to over middle of distance between pelvic-fin base and pectoral-fin origin; patches of breast scales commonly connected by scattered scales or forming 1-3 regular rows of scales; total vertebrae commonly 38-39 (22 + 17, 22 + 16 or 21 + 17). The study reveals unrecognised morphological diversity within the group and provides the groundwork for future taxonomic study in Phoxinus in the Adriatic region and the Danube Basin.

A story of nasal horns: two new subspecies of Iguana Laurenti, 1768 (Squamata, Iguanidae) in Saint Lucia, St Vincent amp; the Grenadines, and Grenada (southern Lesser Antilles).

The Lesser Antilles, in the Eastern Caribbean, were long considered to have only two species in the genus Iguana Laurenti 1768: the Lesser Antillean iguana Iguana delicatissima, which is endemic to parts of the Lesser Antilles, and the Common green iguana Iguana iguana, which also occurs throughout Central and South America. No subspecies are currently recognised. However, herpetologists and reptile collectors have pointed out strong physical differences between some of the island populations of Iguana iguana and those from the continent. Drawing on both morphological and genetic data, this paper describes two subspecies of the Common green iguana Iguana iguana from the southern Lesser Antilles, specifically the countries of Saint Lucia Iguana iguana sanctaluciae and Iguana iguana insularis from St Vincent the Grenadines, and Grenada. The form on the island of Saint Vincent has not been identified. The new subspecies are described based on the following unique combination of characters: Presence of high median and medium to small lateral horns on the snout; Small subtympanic plate not exceeding 20% of the eardrum size; Two or three scales of decreasing size anterior to the subtympanic plate; Fewer than ten small to medium triangular gular spikes; Medium sized dewlap; Low number of small to medium dispersed nuchal tubercles; Dark brown iris, with the white of the eye visible; Oval, prominent nostril; Short and relatively flat head; High dorsal spines; No swelling of the jowls in reproductively active males.                Iguana iguana sanctaluciae has in adults vertical black stripes on body and tail and a black dewlap whereas Iguana iguana insularis is pale grey or creamy white in adults.                Both subspecies are globally threatened by unsustainable hunting (including the pet trade) and by invasive alien species,     including hybridization from invasive iguanas from South and Central America (I. iguana iguana and I. rhinolopha, considered here as full species) that have become established in all three countries. The authors call for stronger measures to conserve the remaining purebred Iguana i. insularis and Iguana i. sanctaluciae ssp. nov. throughout their ranges and for further research to identify other cryptic species and subspecies of Iguana in the Lesser Antilles.

Distribution of endangered Italian gudgeon Romanogobio benacensis (Cypriniformes, Cyprinidae, Gobioninae) with remarks on distinguishing morphological characters.

Distribution data on many freshwater fish species in Croatia are scarce and species identifications are difficult, requiring further detailed studies. This paper presents a report of the Italian gudgeon Romanogobio benacensis from the Mirna River in the Istra Peninsula in Croatia, in the south-east from its previously known distribution range. The identification of R. benacensis in Croatia was supported by a morphological comparison with R. benacensis from Italy and Slovenia, the common gudgeon Gobio gobio, and the Danubian gudgeon Gobio obtusirostris from geographically close locations. A combination of character states (number of scales between anus and anal-fin origin, branched pectoral-fin rays, lateral-line scales, total, abdominal, and caudal vertebrae, and the size and number of lateral blotches) distinguishes R. benacensis from both G. gobio and G. obtusirostris. The phylogenetic analyses using mitochondrial sequences of cytochrome b gene confirmed that specimens from the Mirna River belong to R. benacensis. Also, Reka River system (Adriatic Sea basin) in Slovenia is inhabited by a possibly introduced Danubian gudgeon, G. obtusirostris, and not by R. benacensis.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.

Prevalence of the pathogen Aphanomyces astaci in freshwater crayfish populations in Croatia.

The Oomycete Aphanomyces astaci is an obligate crayfish parasite that co-evolved with American crayfish species, and they therefore generally live in a balanced relationship. On the contrary, European native crayfish are highly susceptible to A. astaci, and infestation with it causes development of the lethal disease termed crayfish plague. Until now, 5 A. astaci strains have been described from the freshwater crayfish present in Europe. In this study we aimed to investigate the occurrence of the pathogen A. astaci in Croatian native and non-native crayfish populations, as well as to genotype established strains using microsatellite markers and obtain information on the pathogen's epidemiology. Our results showed that the pathogen is widespread in both native and non-native crayfish populations. Agent level, when positive, in non-native crayfish was generally low; in native species it was higher. Genotyping from microsatellites proved the presence of the B (Ps) strain in non-native species (Pacifastacus leniusculus), while the A (As) strain was detected from viable native species (Astacus astacus and Austropotamobius torrentium) that are distributed in areas lacking non-native crayfish. The genotype from A. torrentium differed from a typical A (As) by 1 allele. Strain B (Ps) was identified in native Astacus leptodactylus from the population that co-occurs with P. leniuscuslus. Interestingly, in 1 A. leptodactylus population both A (As) and B (Ps) strains were present.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.