Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium

1. This study examines phylogeography and phylogeny of the threatened stone crayfish, Austropotamobius torrentium, in order to elucidate the role of the Dinaric Karst geology in shaping the evolutionary history and genetic diversity of aquatic fauna in the western Balkans. Mitochondrial 16S rRNA and COI genes were partially sequenced from 188 and 159 crayfish, respectively, sampled from 70 localities. Phylogenetic relationships were reconstructed using four methods of phylogenetic inference. Divergence times between phylogroups were estimated in a Bayesian framework, and their demographic history was examined using neutrality tests and mismatch distribution analysis. 2. Seven geographically localised phylogroups separated by pronounced genetic gaps were found. Five of them have a distribution range within the northern‐central Dinaric (NCD) region, while the remaining two include populations from the southern Balkans (SB) and central and south‐eastern Europe (CSE). The oldest divergence event separated two NCD lineages from the rest of populations in the Late Miocene or Early Pliocene. Divergences amongst the five NCD phylogroups and SB + CSE occurred in the Pliocene. The most recent split separated SB and CSE phylogroups during the Late Pliocene. For both genes, uncorrected pairwise divergences between most of the phylogroups (4.1–8.7% for COI and 1.6–4.8% for 16S rRNA) were of the same range as, or higher than, some of the interspecific distances previously reported for the genus Austropotamobius. 3. Geographically isolated and deeply divergent cryptic monophyletic phylogroups within A. torrentium in the NCD region arose in the course of intensification of Neotectonic movements during the Pliocene and the beginning of the Pleistocene and the development of karstification that has heavily fragmented the palaeohydrography of the area. The results confirm a gradual north–south expansion of stone crayfish during the pre‐Pleistocene that preceded the rapid northward post‐glacial re/colonisation of central Europe (CSE phylogroup) through the Danube drainage. 4. Austropotamobius torrentium comprises morphologically cryptic but molecularly distinct taxa. Considering the relatively small geographical areas they inhabit, the NCD phylogroups of stone crayfish should be given the highest conservation priority.     

Gondwanan radiation of the Southern Hemisphere crayfishes (Decapoda: Parastacidae): evidence from fossils and molecules

Aim The sequential break‐up of Gondwana is thought to be a dominant process in the establishment of shared biota across landmasses of the Southern Hemisphere. Yet similar distributions are shared by taxa whose radiations clearly post‐date the Gondwanan break‐up. Thus, determining the contribution of vicariance versus dispersal to seemingly Gondwanan biota is complex. The southern freshwater crayfishes (family Parastacidae) are distributed on Australia and New Guinea, South America, Madagascar and New Zealand and are unlikely to have dispersed via oceans, owing to strict freshwater limitations. We test the hypotheses that the break‐up of Gondwana has led to (1) a predominately east–west (((Australia, New Zealand: 80 Ma) Madagascar: 160–121 Ma) South America: 165–140 Ma), or (2) a southern (((Australia, South America: 52–35 Ma) New Zealand: 80 Ma) Madagascar: 160–121 Ma) pattern for parastacid crayfish. Further, we examine the evidence for a complete drowning of New Zealand and subsequent colonization by freshwater crayfish. Location Southern Hemisphere. Methods The evolutionary relationships among the 15 genera of Parastacidae were reconstructed using mitochondrial [16S, cytochrome c oxidase subunit I (COI)] and nuclear (18S, 28S) sequence data and maximum likelihood and Bayesian methods of phylogenetic reconstruction. A Bayesian (multidivtime ) molecular dating method using six fossil calibrations and phylogenetic inference was used to estimate divergence time among crayfish clades on Gondwanan landmasses. Results The South American crayfish are monophyletic and a sister group to all other southern crayfish. Australian crayfish are not monophyletic, with two Tasmanian genera, Spinastacoides and Ombrastacoides, forming a clade with New Zealand and Malagasy crayfish (both monophyletic). Divergence of crayfish among southern landmasses is estimated to have occurred around the Late Jurassic to Early Cretaceous (109–178 Ma). Main conclusions The estimated phylogenetic relationships and time of divergence among the Southern Hemisphere crayfishes were consistent with an east–west pattern of Gondwanan divergence. The divergence between Australia and New Zealand (109–160 Ma) pre‐dated the rifting at around 80 Ma, suggesting that these lineages were established prior to the break‐up. Owing to the age of the New Zealand crayfish, we reject the hypothesis that there was a complete drowning of New Zealand crayfish habitat.     

Modern Northern Domestic Horses Carry Mitochondrial DNA Similar to Przewalski’s Horse

Several recent studies have suggested past gene flow between the Przewalski’s horse and modern domestic horse and questioned the wild origin of the Przewalski’s horse. Mitochondrial DNA has placed representatives of the Przewalski’s horse into three among the eighteen haplogroups detected from the modern horse. Of these, two haplogroups have so far been found exclusively in the Przewalski’s horse, while the one shared with the domestic horse includes captive individuals that have uncertain pedigrees. We recently found five domestic horse individuals of North European horse breeds to carry a mitochondrial haplogroup that was previously confined only to the Przewalski’s horse. These individuals were sequenced for 6039 bp of mitochondrial DNA and used, together with domestic and Przewalski’s horse sequences presenting all horse haplogroups, to examine the phylogenetic relationships and to date the divergence time between Przewalski’s and domestic horse clusters within this haplogroup. The divergence was dated to have likely occurred about 13,300–11,400 years ago, which coincides with the time of the Younger Dryas.

     

The biogeography and phylogeny of schizothoracine fishes (Schizopygopsis) in the Qinghai‐Tibetan Plateau

Freshwater fish belonging to the genus Schizopygopsis are widespread in drainages throughout the Qinghai‐Tibetan Plateau and, thus, a model group with which to investigate how paleo‐drainage changes linked to historical uplifting within the Qinghai‐Tibetan Plateau influence speciation. To date, the phylogenetic and taxonomic relationships within Schizopygopsis remain controversial. In this study, we constructed a comprehensive molecular phylogeny of Schizopygopsis based on six mitochondrial gene sequences. We compared the taxonomic relationships revealed by this phylogeny with those obtained from morphological data. We also used this phylogeny to assess the extent to which the evolution of Schizopygopsis has been driven by paleo‐drainage changes linked to uplifting of the Qinghai‐Tibetan Plateau. Results indicated that all Schizopygopsis taxa formed a monophyletic group comprising five major clades, which were inconsistent with the taxonomic relationships based on morphology for this group. Our results also strongly supported the validity of S. anteroventris and S. microcephalus as distinct species within Schizopygopsis. Molecular calibrations showed that species within the middle Yangtze species diverged earlier (~4.5 Mya) than species within the Indus River (~3.0 Mya), the Mekong River (~2.8 Mya) and the Tsangpo + Salween rivers (~2.5 Mya). The most recent evolutionary splits occurred among species from the upper and lower Yangtze River, the Yellow River and the Qiadam Basin at about 1.8 to 0.3 Mya. Our molecular evidence and use of the molecular clock calibration have allowed us to associate speciation events within the genus Schizopygopsis to the formation and separation of paleo‐drainage connections caused by tectonic events during the uplifting of the Qinghai‐Tibetan Plateau (~4.5 Mya). This work underlines the dominant role of vicariance in shaping the evolutionary history of the genus Schizopygopsis. Further research using multiple loci and more extensive sampling will reveal a more complete picture of the phylogenetic relationships and biogeography of Schizopygopsis fishes.     

Evolutionary history of the scorpionfly Dicerapanorpa magna (Mecoptera,Panorpidae)

Climate changes can have fundamental impacts on the distributional patterns of montane species, and range shifts frequently lead to allopatric divergence followed by the establishment of secondary contact zones. Many European and North American organisms have retreated to southern refugia during glacial periods and colonized northward during postglacial periods, but little is known about the evolutionary response of cold‐adapted insects to Pleistocene climate changes in eastern Asia. The scorpionfly Dicerapanorpa magna (Chou), with cold temperate habitat preference and weak dispersal ability, provides a good model system to explore how climate changes have influenced the distribution and divergence of cold‐adapted insects in eastern Asia. This study reconstructed the demographic dynamics and evolutionary history of D. magna with phylogeographic approaches, and predicted the species’ suitable areas under the Last Glacial Maximum (LGM) and current scenarios with the ecological niche modelling analysis. The mitochondrial cytochrome c oxidase subunit I resolved three phylogenetic lineages in D. magna dating back to Pleistocene, corresponding well with the geographically isolated Qinling, Bashan and Minshan Mountains. The ecological niche modelling recovered the suitable habitats for D. magna were the Qinling and Bashan Mountains under LGM and current conditions. The three lineages of D. magna might be in a process of incipient speciation, and likely derived their current distribution from separate glacial origins, followed by vicariance and divergence.     

Evolutionary divergence in Dolichopoda cave crickets: A comparison of single copy DNA hybridization data with allozymes and morphometric distances

In this paper we attempt to investigate relationships between the amount of genetic divergence in nuclear genes and the degree of morphological differentiation for different sets of characters in Dolichopoda cave crickets. Six populations representing five Dolichopoda species from Central and Southern Italy have been studied. The overall genetic divergence at nuclear genes was estimated both by single copy DNA-DNA hybridization and allozyme frequencies at 26 loci. Euclidean distances for two multivariate sets of morphometric variables: one describing body and appendage morphology, the other male epiphallus shape. Results showed a close agreement between the branching patterns of ΔTm values from DNA hybridization and Nei's allozyme distance values. On the other hand, patterns of morphological divergence revealed independent trends, although the branching pattern based on epiphallus morphology matched to some extent the phylogenies inferred from molecular data. The relative value of molecular and morphological characters as reliable phylogenetic tracers was evaluated in relation to their dependence on evolutionary factors. Implications of these findings on the calibration of molecular clocks are also discussed. The absolute rate of molecular change based on scDNA was estimated to be at least 0.98% divergence/my/lineage. This result is in agreement with calibrations attempted on other insects. Estimates of time of divergence based on allozymes (Nei's D) were highly consistent with the estimate from geological data.     

Habitat and shelter requirements of the stone crayfish,Austropotamobius torrentium Schrank

The attractiveness of shelters and the relationship between crayfish size and refuge size were investigated in the stone crayfish Austropotamobius torrentium. Shelter suitability was mainly determined by water velocity, stone surface area and stone width. Water depth and stone height had no influence on crayfish occupancy of stones. Austropotamobius torrentium avoided shelters in areas of water velocities exceeding 25 cm s^–1. They preferred large stones with a bearing surface greater than 300 cm². The probability of crayfish occupation rose quickly as stones' bearing surface increased up to 900 cm². Crayfish were exclusively caught underneath stones at least 3.19 times longer and 1.25 times wider than the carapace length of the refuge occupant. Large males occupied larger stones, where no relationship was detected between female size and stone refuge size.     

Complex longitudinal diversification across South China and Vietnam in Stejneger's pit viper,Viridovipera stejnegeri (Schmidt, 1925) (Reptilia: Serpentes: Viperidae)

Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well‐supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan–Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west–east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate‐mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon.     

Molecular phylogeny and zoogeography of the freshwater crayfish genus Cherax Erichson (Decapoda: Parastacidae) in Australia

The evolutionary history and biogeography of freshwater-dependent taxa in Australia is of intrinsic interest given the present-day aridity of this continent. Cherax is the most widespread and one of the most species-rich of Australia's nine freshwater crayfish genera. The phylogenetic relationships amongst 19 of the 23 Australian Cherax were established from mitochondrial DNA sequences representing the 12S rRNA and 16S rRNA gene regions. The relationships among species support an initial east–west separation, followed by a north–south divergence in eastern Australia. Molecular clock estimations suggest that these divergences date back to the Miocene. The phylogenetic relationships support endemic speciation within geographical regions and indicate that long-distance dispersal has not led to recent speciation as previously hypothesized. This new evolutionary scenario is consistent with the climatic history of Australia and the evolutionary history of other similarly distributed freshwater-dependent organisms in Australia.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 553–563.     

Integration of Bayesian molecular clock methods and fossil-based soft bounds reveals early Cenozoic origin of African lacertid lizards

Background  

Although current molecular clock methods offer greater flexibility in modelling evolutionary events, calibration of the clock with dates from the fossil record is still problematic for many groups. Here we implement several new approaches in molecular dating to estimate the evolutionary ages of Lacertidae, an Old World family of lizards with a poor fossil record and uncertain phylogeny. Four different models of rate variation are tested in a new program for Bayesian phylogenetic analysis called TreeTime, based on a combination of mitochondrial and nuclear gene sequences. We incorporate paleontological uncertainty into divergence estimates by expressing multiple calibration dates as a range of probabilistic distributions. We also test the reliability of our proposed calibrations by exploring effects of individual priors on posterior estimates.     

Calibration age and quartet divergence date estimation

The date of a single divergence point--between living alligators and crocodiles--was estimated with quartet dating using calibrations of widely divergent ages. For five mitochondrial sequence datasets, there is a clear relationship between calibration age and quartet estimate--quartets based on two relatively recent calibrations support younger divergence estimates than do quartets based on two older calibrations. Some of the estimates supported by young quartets are impossibly young and exclude the first appearance of the group in the fossil record as too old. The older estimates--those based on two relatively old calibrations--may be overestimates, and those based on one old and one recent calibration support divergence estimates very close to fossil data. This suggests that quartet dating methods may be most effective when calibrations are applied from different parts of a clade's history.     

Loss of genetic diversity means loss of geological information: the endangered Japanese crayfish exhibits remarkable historical footprints

Intra-specific genetic diversity is important not only because it influences population persistence and evolutionary potential, but also because it contains past geological, climatic and environmental information. In this paper, we show unusually clear genetic structure of the endangered Japanese crayfish that, as a sedentary species, provides many insights into lesser-known past environments in northern Japan. Over the native range, most populations consisted of unique 16S mtDNA haplotypes, resulting in significant genetic divergence (overall F _ST = 0.96). Owing to the simple and clear structure, a new graphic approach unraveled a detailed evolutionary history; regional crayfish populations were comprised of two distinct lineages that had experienced contrasting demographic processes (i.e. rapid expansion vs. slow stepwise range expansion) following differential drainage topologies and past climate events. Nuclear DNA sequences also showed deep separation between the lineages. Current ocean barriers to dispersal did not significantly affect the genetic structure of the freshwater crayfish, indicating the formation of relatively recent land bridges. This study provides one of the best examples of how phylogeographic analysis can unravel a detailed evolutionary history of a species and how this history contributes to the understanding of the past environment in the region. Ongoing local extinctions of the crayfish lead not only to loss of biodiversity but also to the loss of a significant information regarding past geological and climatic events.     

Comparative ultrastructure of the spermatozoa of three crayfish species: Austropotamobius torrentium,Pacifastacus leniusculus,and Astacus astacus (Decapoda: Astacidae)

This study reports about the spermatozoal ultrastructure of three species of astacid crayfish, i.e., the stone crayfish Austropotamobius torrentium, signal crayfish Pacifastacus leniusculus, and noble crayfish Astacus astacus. The acrosome is a cup shaped and electron‐dense structure at the anterior of the spermatozoon and comprises three layers of differing electron densities filled with parallel filaments that extend from the base to the apical zone. The acrosome was significantly longer in A. astacus than in P. leniusculus and the shortest acrosome belongs to A. torrentium. The width of the acrosome was significantly narrower in A. torrentium than in P. leniusculus and the widest acrosome belongs to A. astacus. The L:W ratio was significantly greater in A. torrentium than in P. leniusculus and the lowest ratio belongs to A. astacus. Radial arms are visible on each side of the acrosome or nucleus in sagittal view and wrap around the spermatozoon. Each radial arm comprises a parallel bundle of microtubules arranged along the long axis within a sheath. The nucleus, with decondensed material, is located in the posterior of the cell. All parts of the spermatozoon are tightly enclosed within an extracellular capsule. Despite a well‐conserved general structure and similarity of pattern among these spermatozoa, differences in the dimensions of the acrosome within the studied species may be useful to help distinguish the different crayfish species. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Phylogeography and Pleistocene refugia of the Little Owl Athene noctua inferred from mtDNA sequence data

Pleistocene glaciations greatly affected the distribution of genetic diversity in animal populations. The Little Owl is widely distributed in temperate regions and could have survived the last glaciations in southern refugia. To describe the phylogeographical structure of European populations, we sequenced the mitochondrial cytochrome c oxidase I (COI) and control region (CR1) in 326 individuals sampled from 22 locations. Phylogenetic analyses of COI identified two deeply divergent clades: a western haplogroup distributed in western and northwestern Europe, and an eastern haplogroup distributed in southeastern Europe. Faster evolving CR1 sequences supported the divergence between these two main clades, and identified three subgroups within the eastern clade: Balkan, southern Italian and Sardinian. Divergence times estimated from COI with fossil calibrations indicate that the western and eastern haplogroups split 2.01–1.71 Mya. Slightly different times for splits were found using the standard 2% rate and 7.3% mtDNA neutral substitution rate. CR1 sequences dated the origin of endemic Sardinian haplotypes at 1.04–0.26 Mya and the split between southern Italian and Balkan haplogroups at 0.72–0.21 Mya, coincident with the onset of two Pleistocene glaciations. Admixture of mtDNA haplotypes was detected in northern Italy and in central Europe. These findings support a model of southern Mediterranean and Balkan refugia, with postglacial expansion and secondary contacts for Little Owl populations. Central and northern Europe was predominantly recolonized by Little Owls from Iberia, whereas expansion out of the Balkans was more limited. Northward expansion of the Italian haplogroup was probably prevented by the Alps, and the Sardinian haplotypes remained confined to the island. Results showed a clear genetic pattern differentiating putative subspecies. Genetic distances between haplogroups were comparable with those recorded between different avian species.     

Correlating Bayesian date estimates with climatic events and domestication using a bovine case study

The tribe Bovini contains a number of commercially and culturally important species, such as cattle. Understanding their evolutionary time scale is important for distinguishing between post-glacial and domestication-associated population expansions, but estimates of bovine divergence times have been hindered by a lack of reliable calibration points. We present a Bayesian phylogenetic analysis of 481 mitochondrial D-loop sequences, including 228 radiocarbon-dated ancient DNA sequences, using a multi-demographic coalescent model. By employing the radiocarbon dates as internal calibrations, we co-estimate the bovine phylogeny and divergence times in a relaxed-clock framework. The analysis yields evidence for significant population expansions in both taurine and zebu cattle, European aurochs and yak clades. The divergence age estimates support domestication-associated expansion times (less than 12 kyr) for the major haplogroups of cattle. We compare the molecular and palaeontological estimates for the Bison-Bos divergence.     

Error in Estimation of Rate and Time Inferred from the Early Amniote Fossil Record and Avian Molecular Clocks

The best reconstructions of the history of life will use both molecular time estimates and fossil data. Errors in molecular rate estimation typically are unaccounted for and no attempts have been made to quantify this uncertainty comprehensively. Here, focus is primarily on fossil calibration error because this error is least well understood and nearly universally disregarded. Our quantification of errors in the synapsid–diapsid calibration illustrates that although some error can derive from geological dating of sedimentary rocks, the absence of good stem fossils makes phylogenetic error the most critical. We therefore propose the use of calibration ages that are based on the first undisputed synapsid and diapsid. This approach yields minimum age estimates and standard errors of 306.1±8.5 MYR for the divergence leading to birds and mammals. Because this upper bound overlaps with the recent use of 310 MYR, we do not support the notion that several metazoan divergence times are significantly overestimated because of serious miscalibration (sensu Lee 1999). However, the propagation of relevant errors reduces the statistical significance of the pre-K–T boundary diversification of many bird lineages despite retaining similar point time estimates. Our results demand renewed investigation into suitable loci and fossil calibrations for constructing evolutionary timescales.[Reviewing Editor: Martin Kreitman]     

Assessing the quality of molecular divergence time estimates by fossil calibrations and fossil-based model selection

Estimates of species divergence times using DNA sequence data are playing an increasingly important role in studies of evolution, ecology and biogeography. Most work has centred on obtaining appropriate kinds of data and developing optimal estimation procedures, whereas somewhat less attention has focused on the calibration of divergences using fossils. Case studies with multiple fossil calibration points provide important opportunities to examine the divergence time estimation problem in new ways. We discuss two cross-validation procedures that address different aspects of inference in divergence time estimation. 'Fossil cross-validation' is a procedure used to identify the impact of different individual calibrations on overall estimation. This can identify fossils that have an exceptionally large error effect and may warrant further scrutiny. 'Fossil-based model cross-validation' is an entirely different procedure that uses fossils to identify the optimal model of molecular evolution in the context of rate smoothing or other inference methods. Both procedures were applied to two recent studies: an analysis of monocot angiosperms with eight fossil calibrations and an analysis of placental mammals with nine fossil calibrations. In each case, fossil calibrations could be ranked from most to least influential, and in one of the two studies, the fossils provided decisive evidence about the optimal molecular evolutionary model.     

Comparison of likelihood and Bayesian methods for estimating divergence times using multiple gene Loci and calibration points, with application to a radiation of cute-looking mouse lemur species

Divergence time and substitution rate are seriously confounded in phylogenetic analysis, making it difficult to estimate divergence times when the molecular clock (rate constancy among lineages) is violated. This problem can be alleviated to some extent by analyzing multiple gene loci simultaneously and by using multiple calibration points. While different genes may have different patterns of evolutionary rate change, they share the same divergence times. Indeed, the fact that each gene may violate the molecular clock differently leads to the advantage of simultaneous analysis of multiple loci. Multiple calibration points provide the means for characterizing the local evolutionary rates on the phylogeny. In this paper, we extend previous likelihood models of local molecular clock for estimating species divergence times to accommodate multiple calibration points and multiple genes. Heterogeneity among different genes in evolutionary rate and in substitution process is accounted for by the models. We apply the likelihood models to analyze two mitochondrial protein-coding genes, cytochrome oxidase II and cytochrome b, to estimate divergence times of Malagasy mouse lemurs and related outgroups. The likelihood method is compared with the Bayes method of Thorne et al. (1998, Mol. Biol. Evol. 15:1647-1657), which uses a probabilistic model to describe the change in evolutionary rate over time and uses the Markov chain Monte Carlo procedure to derive the posterior distribution of rates and times. Our likelihood implementation has the drawbacks of failing to accommodate uncertainties in fossil calibrations and of requiring the researcher to classify branches on the tree into different rate groups. Both problems are avoided in the Bayes method. Despite the differences in the two methods, however, data partitions and model assumptions had the greatest impact on date estimation. The three codon positions have very different substitution rates and evolutionary dynamics, and assumptions in the substitution model affect date estimation in both likelihood and Bayes analyses. The results demonstrate that the separate analysis is unreliable, with dates variable among codon positions and between methods, and that the combined analysis is much more reliable. When the three codon positions were analyzed simultaneously under the most realistic models using all available calibration information, the two methods produced similar results. The divergence of the mouse lemurs is dated to be around 7-10 million years ago, indicating a surprisingly early species radiation for such a morphologically uniform group of primates.     

Associations between stream habitat characteristics and native and alien crayfish occurrence

Human mediated introductions of non-indigenous crayfish species (NICS) are responsible for their rapid colonisation of European freshwaters. The introduction of North American crayfish is furthermore linked to the spread of crayfish plague and the decline of indigenous crayfish species (ICS). As the management of ICS and NICS have become necessary, a detailed knowledge on their distribution and ecological requirements is needed. We studied the current range of native noble crayfish Astacus astacus and stone crayfish Austropotamobius torrentium, as well as alien signal crayfish Pacifastacus leniusculus in Carinthia (Austria) and evaluated environmental and physical habitat features in streams with and without crayfish. Meanwhile, the loss of many ICS populations was recorded and alien P. leniusculus was found to be widespread in this region. Most of the habitat features of streams having crayfish differed significantly from sites lacking crayfish for at least one investigated native or alien species. Furthermore, multivariate and regression analyses showed specific differences in the habitat use of the investigated crayfish. Our results showed that the presence of alien P. leniusculus was associated with larger and smoother sloped lowland rivers, while the occurrence of the two native species was confined to smaller streams either at higher altitudes and with distinct physical habitat conditions (A. torrentium) or with moderate water temperatures (A. astacus). This study helps to identify potential refuge areas for the endangered native species and to predict the further spread of the most common non-native crayfish species in European streams.     

The effect of inappropriate calibration: three case studies in molecular ecology

Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time-scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events.