Modern morphological methods for tadpole studies. A comparison of micro-CT, and clearing and staining protocols modified for frog larvae.

Micro-computed tomography (micro-CT) scanning and three-dimensional reconstruction have revolutionized morphological studies. Whereas species descriptions and comparative studies formerly were based on external appearance and dissection, we now can visualize muscles, skeleton and viscera in intact animals. In most cases, visualization of internal structures depends on appropriate staining methods. Staining with iodine, phosphotungstic acid (PTA) and osmium tetroxide are established methods, but some problems remain. Agents like osmium tetroxide are toxic and the contrast of cartilage generally is unsatisfactory with osmium tetroxide, iodine or PTA. Furthermore, staining results vary for different animals and different developmental stages. We investigated critical point drying as an inexpensive, nontoxic and rapid alternative to staining for frog tadpoles. Critical point drying enables visualization of cartilage and its differentiation from muscle tissue. Shrinkage generally is acceptable. We also present a protocol for clearing and staining frog tadpoles.

Comprehensive DNA barcoding of the herpetofauna of Germany.

We present the first comprehensive DNA barcoding study of German reptiles and amphibians representing likewise the first on the European herpetofauna. A total of 248 barcodes for all native species and subspecies in the country and a few additional taxa were obtained in the framework of the projects 'Barcoding Fauna Bavarica' (BFB) and 'German Barcode of Life' (GBOL). In contrast to many invertebrate groups, the success rate of the identification of mitochondrial lineages representing species via DNA barcode was almost 100% because no cases of Barcode Index Number (BIN) sharing were detected within German native reptiles and amphibians. However, as expected, a reliable identification of the hybridogenetic species complex in the frog genus Pelophylax was not possible. Deep conspecific lineages resulting in the identification of more than one BIN were found in Lissotriton vulgaris, Natrix natrix and the hybridogenetic Pelophylax complex. A high variety of lineages with different BINs was also found in the barcodes of wall lizards (Podarcis muralis), confirming the existence of many introduced lineages and the frequent occurrence of multiple introductions. Besides the reliable species identification of all life stages and even of tissue remains, our study highlights other potential applications of DNA barcoding concerning German amphibians and reptiles, such as the detection of allochthonous lineages, monitoring of gene flow and also noninvasive sampling via environmental DNA. DNA barcoding based on COI has now proven to be a reliable and efficient tool for studying most amphibians and reptiles as it is already for many other organism groups in zoology.

Population signatures of large-scale, long-term disjunction and small-scale, short-term habitat fragmentation in an Afromontane forest bird.

The Eastern Afromontane cloud forests occur as geographically distinct mountain exclaves. The conditions of these forests range from large to small and from fairly intact to strongly degraded. For this study, we sampled individuals of the forest bird species, the Montane White-eye Zosterops poliogaster from 16 sites and four mountain archipelagos. We analysed 12 polymorphic microsatellites and three phenotypic traits, and calculated Species Distribution Models (SDMs) to project past distributions and predict potential future range shifts under a scenario of climate warming. We found well-supported genetic and morphologic clusters corresponding to the mountain ranges where populations were sampled, with 43% of all alleles being restricted to single mountains. Our data suggest that large-scale and long-term geographic isolation on mountain islands caused genetically and morphologically distinct population clusters in Z. poliogaster. However, major genetic and biometric splits were not correlated to the geographic distances among populations. This heterogeneous pattern can be explained by past climatic shifts, as highlighted by our SDM projections. Anthropogenically fragmented populations showed lower genetic diversity and a lower mean body mass, possibly in response to suboptimal habitat conditions. On the basis of these findings and the results from our SDM analysis we predict further loss of genotypic and phenotypic uniqueness in the wake of climate change, due to the contraction of the species' climatic niche and subsequent decline in population size.

Species distribution models contribute to determine the effect of climate and interspecific interactions in moving hybrid zones.

Climate is a major factor delimiting species' distributions. However, biotic interactions may also be prominent in shaping geographical ranges, especially for parapatric species forming hybrid zones. Determining the relative effect of each factor and their interaction of the contact zone location has been difficult due to the lack of broad scale environmental data. Recent developments in species distribution modelling (SDM) now allow disentangling the relative contributions of climate and species' interactions in hybrid zones and their responses to future climate change. We investigated the moving hybrid zone between the breeding ranges of two parapatric passerines in Europe. We conducted SDMs representing the climatic conditions during the breeding season. Our results show a large mismatch between the realized and potential distributions of the two species, suggesting that interspecific interactions, not climate, account for the present location of the contact zone. The SDM scenarios show that the southerly distributed species, Hippolais polyglotta, might lose large parts of its southern distribution under climate change, but a similar gain of novel habitat along the hybrid zone seems unlikely, because interactions with the other species (H. icterina) constrain its range expansion. Thus, whenever biotic interactions limit range expansion, species may become 'trapped' if range loss due to climate change is faster than the movement of the contact zone. An increasing number of moving hybrid zones are being reported, but the proximate causes of movement often remain unclear. In a global context of climate change, we call for more interest in their interactions with climate change.

Taxonomy of the super-cryptic Hyperolius nasutus group of long reed frogs of Africa (Anura: Hyperoliidae), with descriptions of six new species.

Specimens from across the range of the Hyperolius nasutus species group were sequenced for two mitochondrial genes and one nuclear gene. Advertisement calls were recorded from the same specimens where possible, and morphological characters were compared. Bayesian inference and maximum likelihood produced a tree indicating 16 clades. The clades show little or no overlap in combinations of 16S sequence difference, shared tyr haplotypes, advertisement call parameters, snout profiles and webbing. On the basis of these data we recognise H. acuticeps, H. adspersus, H. benguellensis, H. dartevellei, H. igbettensis, H. nasutus, H. nasicus, H. poweri, H. viridis and describe six new species: Hyperolius friedemanni sp. nov. Mercurio & Rödel, Hyperolius howelli sp. nov. Du Preez & Channing, Hyperolius inyangae sp. nov. Channing, Hyperolious jacobseni sp. nov. Channing, Hyperolius rwandae sp. nov. Dehling, Sinsch, R6del & Channing, and Hyperolius lupiroensis sp. nov. Channing. Hyperolius lamottei is confirmed to be outside the H. nasutus group clade. Hyperolius granulatus, H. oxyrhynchus, H. punctulatus and H. sagitta are assigned as junior synonyms. As our results are based on a small number of specimens, these hypotheses await testing with larger sample sizes and more characters. A species distribution model suggests where outlier populations might be found.

[Pain management and coping behaviour in premature babies (< 1500 g) at corrected age of 36 months influence of neonatal experience and maternal anxiety]. / Schmerzverhalten und Bewältigungsformen von Frühgeborenen (< 1500g) im korrigierten Alter von 36 Monaten -- Einflüsse neonataler Erfahrungen und mütterlicher Angste.

INTRODUCTION: Neonatal experience of pain and distress can lead to developmental problems, which can be associated with long-term emotional and behavioural disorders. The aim of the study was to analyse the effects of early experiences of pain and maternal reactions on the pain and coping behaviour of preterm infants. METHODS: In a prospective longitudinal study of 69 very low birth weight (VLBW) preterm infants, neonatal data regarding painful manipulations, analgesics and sedatives, and general medical condition (Nursery Neurobiological Risk Score; NBRS) were assessed. At the (corrected) age of 36 months, 53 preterm infants and a control group of 23 full-term infants were re-examined. Pain and coping behaviour were estimated by a questionnaire. Maternal anxiety was assessed in semi-structured interviews at the age of 3, 12 and 36 months in the preterm group. RESULTS: The mean gestational age was 29 + 0 weeks (23 + 3 to 34 + 1), the mean birth weight 1058 g (380 to 1480 g) in preterms and 39 + 3 weeks (37 + 0 to 42 + 0) and 3379 g (2400 to 4130 g), respectively, for the full-terms. The sex ratio was equal, 45.3 % of the preterms were multiples (controls 34.8 %). Preterms had higher descriptive scores for all types of pain situations. After controlling for other associated factors, a negative correlation between birth weight and later pain behaviour in medical situations remained. Preterms had a more negative coping behaviour during every day injuries. In terms of coping behaviour, only a shorter inpatient treatment in the neonatal period was associated with social withdrawal after controlling for other associated factors. Maternal anxiety at the age of 12 and 36 months was associated with negative coping behaviour following simple injuries. CONCLUSIONS: While preterms do not have a higher pain threshold in general, a subgroup does have a higher risk for later sensation to pain. Preterms use more unfavourable coping strategies in simple injuries which, in turn, seem to be decisively mediated by maternal anxiety. Future research should focus on psycho-social factors involved in the development of pain reactions, as these can predispose towards behavioural disorders.

[Mother-infant Interactions with Very Low Birth Weight multiple newborns (< 1500 g). A comparison of mother-multiple and mother-single births]. / Mutter-Kind-Interaktion bei sehr leichtgewichtig frühgeborenen Mehrlingen (< 1500 g). Ein Vergleich von Mutter-Mehrlings- und Mutter-Einlings-Dyaden.

BACKGROUND: Assisted reproductive techniques and fertility enhancing therapies have increased the rate of multiple births and, therefore, the risk of prematurity. Our hypothesis is that mothers of preterm multiples are less able to provide such enhancing interactions than mothers of preterm singletons, resulting in a developmental disadvantage for preterm twins and triplets. PATIENTS AND METHODS: Of 77 very low birth weight preterms (VLBW) who were examined prospectively with their mothers in a longitudinal study, 35 were multiples and 42 were singletons. At a corrected age of three months the quality of the mother-infant interaction with multiples vs. singletons was examined. The Mannheim Rating System, a 40-item standardized observation instrument based on a 10 minute videotaped sequence of interaction, was used. RESULTS: The analyses showed several differences between mother-singleton and mother-multiple interactions. Mothers of multiples were less stimulating and reactive and showed less babytalk. Multiple infants were also less reactive than singletons. In mother-multiple dyads there were less verbal exchanges between mother and child. CONCLUSIONS: There are definite differences in mother-multiple compared to mother-singleton interactions, so that VLBW multiples may be at even greater risk for negative mother-infant interactions than singletons.