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The platy limestone deposit of Vallecillo in northeastern Mexico is dated to the early-middle Turonian (Late Cretaceous) and known to contain a variety of well-preserved vertebrate fossils. One of the most common fish species is the teleost Tselfatia formosa. A review of 149 individuals reveals the presence of two types of body shapes (diamond-shaped and torpedo-shaped individuals) which is interpreted as sexual shape dimorphism (SSD). A unimodal size distribution illustrates a dominance of diamond-shaped specimens, but both body shape types are present in small (young) and big sized (old) individuals. The abundance of well-articulated and complete specimens suggests that T. formosa populated deep levels of the water column, which excluded buoyancy and flotation as well as carcass disintegration near the surface. The reconstruction of the dorsal and anal fins suggests the presence of a membrane between each fin ray and allows for ecological comparison of T. formosa with modern fan fishes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Stinnesbeck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although aquatic macroinvertebrates and freshwater fishes are important indicators for freshwater quality assessments, the morphological identification to species-level is often impossible and thus especially in many invertebrate taxa not mandatory during Water Framework Directive monitoring, a pragmatism that potentially leads to information loss. Here, we focus on the freshwater fauna of the River Sieg (Germany) to test congruence and additional value in taxa detection and taxonomic resolution of DNA barcoding vs. morphology-based identification in monitoring routines. Prior generated morphological identifications of juvenile fishes and aquatic macroinvertebrates were directly compared to species assignments using the identification engine of the Barcode of Life Data System. In 18% of the invertebrates morphology allowed only assignments to higher systematic entities, but DNA barcoding lead to species-level assignment. Dissimilarities between the two approaches occurred in 7% of the invertebrates and in 1% of the fishes. The 18 fish species were assigned to 20 molecular barcode index numbers, the 104 aquatic invertebrate taxa to 113 molecular entities. Although the cost-benefit analysis of both methods showed that DNA barcoding is still more expensive (5.30-8.60€ per sample) and time consuming (12.5h), the results emphasize the potential to increase taxonomic resolution and gain a more complete profile of biodiversity, especially in invertebrates. The provided reference DNA barcodes help building the foundation for metabarcoding approaches, which provide faster sample processing and more cost-efficient ecological status determination., Competing Interests: The authors have declared that no competing interests exist.

Lepidodactylus lugubris is a parthenogenetic gecko which has been increasingly expanding its range during the last century. This invasive species has been reported from multiple tropical and subtropical countries in five continents, most of which were colonized in recent times. In order to understand how the realized niche of the species was affected by this dramatic geographic range expansion, we reconstructed the history of the geographic range expansion. We built models of the realized niche of the species at different points in time during the invasion process. This was achieved through the implementation of modern hypervolume construction methods, based on the Hutchinson's niche concept. The models were then compared to detect possible realized climatic niche expansion over time. Furthermore, we investigated possible pathways used by the species to spread. A progressive expansion of the realized niche was identified. As the species spread into new areas, we observed a tendency to colonize regions with warmer temperatures and higher precipitation rates. Finally, we found evidence for cargo shipping being the major pathway through which the species expands its range. Further studies on this topic should aim to investigate the role of biological interactions, and how they shape the distribution of L. lugubris on a global scale. A deeper understanding of this kind of processes will help us tackle the issue of invasive species, which has become a major challenge in conservation biology., Competing Interests: The authors have declared that no competing interests exist.

Habitat demands and species mobility strongly determine the occurrence of species. Sedentary species with specific habitat requirements are assumed to occur more patchy than mobile habitat generalist species, and thus suffer stronger under habitat fragmentation and habitat deterioration. In this study we measured dispersal and habitat preference of three selected butterfly species using mark-release-recapture technique. We used data on species abundance to calculate Species Distribution Models based on high-resolution aerial photographs taken using RGB / NIR cameras mounted on a UAV. We found that microhabitats for species with specific habitat requirements occur spatially restricted. In contrast, suitable habitats are more interconnected and widespread for mobile habitat generalists. Our models indicate that even managed grassland sites have comparatively little habitat quality, while road verges provide high quality micro-habitats. In addition, dispersal was more restricted for specialist butterfly species, and higher for the two other butterfly species with less ecological specialisation. This study shows synergies arising when combining ecological data with high precision aerial pictures and Species Distribution Models, to identify micro-habitats for butterflies. This approach might be suitable to identify and conserve high quality habitats, and to improve nature conservation at the ground., Competing Interests: The authors have declared that no competing interests exist.

The development of modern methods of species delimitation, unified under the "integrated taxonomy" approach, allows a critical examination and re-evaluation of complex taxonomic groups. The rose chafer Protaetia (Potosia) cuprea is a highly polymorphic species group with a large distribution range. Despite its overall commonness, its taxonomy is unclear and subject to conflicting hypotheses, most of which largely fail to account for its evolutionary history. Based on the sequences of two mitochondrial markers from 65 individuals collected across the species range, and a detailed analysis of morphological characters including a geometric morphometry approach, we infer the evolutionary history and phylogeography of the P. cuprea species complex. Our results demonstrate the existence of three separate lineages in the Western Palearctic region, presumably with a species status. However, these lineages are in conflict with current taxonomic concepts. None of the 29 analyzed morphological characters commonly used in the taxonomy of this group proved to be unambiguously species- or subspecies- specific. The geometric morphometry analysis reveals a large overlap in the shape of the analyzed structures (pronotum, meso-metaventral projection, elytra and aedeagus), failing to identify either the genetically detected clades or the classical species entities. Our results question the monophyly of P. cuprea in regard to P. cuprina, as well as the species status of P. metallica. On the other hand, we found support for the species status of the Sicilian P. hypocrita. Collectively, our findings provide a new and original insight into the taxonomy and phylogeny of the P. cuprea species complex. At the same time, the results represent the first attempt to elucidate the phylogeography of these polymorphic beetles.

In order to better understand the complex geologic history of the Mediterranean area, we have analysed evolutionary history, phylogeographic structure and molecular diversity of freshwater fishes belonging to the genus Telestes. As primary freshwater fishes distributed largely in the Mediterranean basin, this genus represents a suitable model system for investigating the historical biogeography of freshwater drainage systems in southern Europe. In this investigation we have included samples representing all Telestes species and based our analyses on one mitochondrial and one nuclear gene. We have investigated phylogenetic structure inside the genus Telestes, estimated divergence times, reconstructed ancestral distribution ranges and described intraspecific molecular diversity. Diversification of Telestes started in the Early Miocene, when the ancestors of T. souffia, lineage comprising T. croaticus and T. fontinalis, and the one comprising T. pleurobipunctatus and T. beoticus got isolated. The remaining species are genetically more closely related and form a common cluster in the recovered phylogenetic trees. Complex geological history of southern Europe, including formation of continental bridges, fragmentation of landmass, closing of the sea corridor, local tectonic activities, led to complicated biogeographical pattern of this genus, caused by multiple colonization events and passovers between ancient rivers and water basins. Especially pronounced diversity of Telestes found in the Adriatic watershed in Croatia and Bosnia and Herzegovina is a consequence of a triple colonization of this area by different lineages, which led to an existence of genetically distinct species in neighboring areas. Significant intraspecific structuring is present in T. souffia, T. muticellus, T. croaticus and T. pleurobipunctatus. Besides in well-structured species, elevated levels of genetic polymorphism were found inside T. turskyi and T. ukliva, as a consequence of their old origin and unconstrained evolutionary history.

Systematic biases such as long branch attraction can mislead commonly relied upon model-based (i.e. maximum likelihood and Bayesian) phylogenetic methods when, as is usually the case with empirical data, there is model misspecification. We present PhyQuart, a new method for evaluating the three possible binary trees for any quartet of taxa. PhyQuart was developed through a process of reciprocal illumination between a priori considerations and the results of extensive simulations. It is based on identification of site-patterns that can be considered to support a particular quartet tree taking into account the Hennigian distinction between apomorphic and plesiomorphic similarity, and employing corrections to the raw observed frequencies of site-patterns that exploit expectations from maximum likelihood estimation. We demonstrate through extensive simulation experiments that, whereas maximum likeilihood estimation performs well in many cases, it can be outperformed by PhyQuart in cases where it fails due to extreme branch length asymmetries producing long-branch attraction artefacts where there is only very minor model misspecification.

Human-mediated biological exchange has had global social and ecological impacts. In sub-Saharan Africa, several domestic and commensal animals were introduced from Asia in the pre-modern period; however, the timing and nature of these introductions remain contentious. One model supports introduction to the eastern African coast after the mid-first millennium CE, while another posits introduction dating back to 3000 BCE. These distinct scenarios have implications for understanding the emergence of long-distance maritime connectivity, and the ecological and economic impacts of introduced species. Resolution of this longstanding debate requires new efforts, given the lack of well-dated fauna from high-precision excavations, and ambiguous osteomorphological identifications. We analysed faunal remains from 22 eastern African sites spanning a wide geographic and chronological range, and applied biomolecular techniques to confirm identifications of two Asian taxa: domestic chicken (Gallus gallus) and black rat (Rattus rattus). Our approach included ancient DNA (aDNA) analysis aided by BLAST-based bioinformatics, Zooarchaeology by Mass Spectrometry (ZooMS) collagen fingerprinting, and direct AMS (accelerator mass spectrometry) radiocarbon dating. Our results support a late, mid-first millennium CE introduction of these species. We discuss the implications of our findings for models of biological exchange, and emphasize the applicability of our approach to tropical areas with poor bone preservation.

The complex climatic and geological history of Southeast Asia has shaped this region's high biodiversity. In particular, sea level fluctuations associated with repeated glacial cycles during the Pleistocene both facilitated, and limited, connectivity between populations. In this study, we used data from two mitochondrial and three anonymous nuclear markers to determine whether a fresh/brackish water killifish, Aplocheilus panchax, Hamilton, 1822, could be used to further understand how climatic oscillations and associated sea level fluctuations have shaped the distribution of biota within this region, and whether such patterns show evidence of isolation within palaeodrainage basins. Our analyses revealed three major mitochondrial clades within A. panchax. The basal divergence of A. panchax mitochondrial lineages was approximately 3.5 Ma, whilst the subsequent divergence timings of these clades occurred early Pleistocene (~2.6 Ma), proceeding through the Pleistocene. Continuous phylogeographic analysis showed a clear west-east dispersal followed by rapid radiation across Southeast Asia. Individuals from Krabi, just north of the Isthmus of Kra, were more closely related to the Indian lineages, providing further evidence for a freshwater faunal disjunction at the Isthmus of Kra biogeographic barrier. Our results suggest that Sulawesi, across the Wallace Line, was colonised relatively recently (~30 ka). Nuclear DNA is less geographically structured, although Mantel tests indicated that nuclear genetic distances were correlated with geographic proximity. Overall, these results imply that recent gene flow, as opposed to historical isolation, has been the key factor determining patterns of nuclear genetic variation in A. panchax, however, some evidence of historical isolation is retained within the mitochondrial genome. Our study further validates the existence of a major biogeographic boundary at the Kra Isthmus, and also demonstrates the use of widely distributed fresh/brackishwater species in phylogeographic studies, and their ability to disperse across major marine barriers in relatively recent time periods.

Neotropical monkey lizards (Polychrus) are arboreal lizards with compressed bodies, partially fused eyelids and strikingly long, whip-like tails. The eight currently recognized species occur in the lowlands of South and Central America. Based on the largest taxon and character sampling to date, we analyze three mitochondrial and one nuclear gene using Bayesian methods to (1) infer the phylogeny of Polychrus under both concatenated-tree and species-tree methods; (2) identify lineages that could represent putative undescribed species; and (3) estimate divergence times. Our species tree places P. acutirostris as the sister taxon to all other species of Polychrus. While the phylogenetic position of P. gutturosus and P. peruvianus is poorly resolved, P. marmoratus and P. femoralis are strongly supported as sister to P. liogaster and P. jacquelinae, respectively. Recognition of P. auduboni and P. marmoratus sensu stricto as distinct species indicates that the populations of "P. marmoratus" from the Amazon and the Atlantic coast in Brazil represent separate species. Similarly, populations of P. femoralis from the Tumbes region might belong to a cryptic undescribed species. Relative divergence times and published age estimates suggest that the orogeny of the Andes did not play a significant role in the early evolution of Polychrus.

The morphology of larvae stages of most amphibians are often completely different than in adults. Tadpole descriptions have historically been based on external characters like morphometrics, color pattern and oral disc structure. Other papers described anatomical details by the use of dissections. The increase in micro-CT scanning technology provides an opportunity to quantify and describe in detail internal characters like skeleton, musculature and organs. To date, no such tadpole descriptions exist for the well-studied Neotropical poison dart frog genus Ranitomeya (Anura: Dendrobatidae). Here we provide descriptions of the internal skeletal, musculature and organ structures of five Ranitomeya species and then provide morphological comparisons. Contrary to previous observations, closely related species display several morphological differences. For example, we observed considerable variation in chondrocranial characters, the extent of cranial ossifications, the appearance of some cranial muscles and the arrangement of inner organs. Further studies on the tadpole morphology of more species of Ranitomeya and other dendrobatid genera are needed to enable us to understand the complete morphological variation in this group.

Malaria is currently one of the world´s major health problems. About a half-million deaths are recorded every year. In Portugal, malaria cases were significantly high until the end of the 1950s but the disease was considered eliminated in 1973. In the past few years, endemic malaria cases have been recorded in some European countries. With the increasing human mobility from countries with endemic malaria to Portugal, there is concern about the resurgence of this disease in the country. Here, we model and map the risk of malaria transmission for mainland Portugal, considering 3 different scenarios of existing imported infections. This risk assessment resulted from entomological studies on An. atroparvus, the only known mosquito capable of transmitting malaria in the study area. We used the malariogenic potential (determined by receptivity, infectivity and vulnerability) applied over geospatial data sets to estimate spatial variation in malaria risk. The results suggest that the risk exists, and the hotspots are concentrated in the northeast region of the country and in the upper and lower Alentejo regions., Competing Interests: The authors have declared that no competing interests exist.

As part of the German Barcode of Life campaign, over 3500 arachnid specimens have been collected and analyzed: ca. 3300 Araneae and 200 Opiliones, belonging to almost 600 species (median: 4 individuals/species). This covers about 60% of the spider fauna and more than 70% of the harvestmen fauna recorded for Germany. The overwhelming majority of species could be readily identified through DNA barcoding: median distances between closest species lay around 9% in spiders and 13% in harvestmen, while in 95% of the cases, intraspecific distances were below 2.5% and 8% respectively, with intraspecific medians at 0.3% and 0.2%. However, almost 20 spider species, most notably in the family Lycosidae, could not be separated through DNA barcoding (although many of them present discrete morphological differences). Conspicuously high interspecific distances were found in even more cases, hinting at cryptic species in some instances. A new program is presented: DiStats calculates the statistics needed to meet DNA barcode release criteria. Furthermore, new generic COI primers useful for a wide range of taxa (also other than arachnids) are introduced., Competing Interests: The authors have declared that no competing interests exist.

By altering or eliminating delicate ecological relationships, non-indigenous species are considered a major threat to biodiversity, as well as a driver of environmental change. Global climate change affects ecosystems and ecological communities, leading to changes in the phenology, geographic ranges, or population abundance of several species. Thus, predicting the impacts of global climate change on the current and future distribution of invasive species is an important subject in macroecological studies. The African clawed frog (Xenopus laevis), native to South Africa, possesses a strong invasion potential and populations have become established in numerous countries across four continents. The global invasion potential of X. laevis was assessed using correlative species distribution models (SDMs). SDMs were computed based on a comprehensive set of occurrence records covering South Africa, North America, South America and Europe and a set of nine environmental predictors. Models were built using both a maximum entropy model and an ensemble approach integrating eight algorithms. The future occurrence probabilities for X. laevis were subsequently computed using bioclimatic variables for 2070 following four different IPCC scenarios. Despite minor differences between the statistical approaches, both SDMs predict the future potential distribution of X. laevis, on a global scale, to decrease across all climate change scenarios. On a continental scale, both SDMs predict decreasing potential distributions in the species' native range in South Africa, as well as in the invaded areas in North and South America, and in Australia where the species has not been introduced. In contrast, both SDMs predict the potential range size to expand in Europe. Our results suggest that all probability classes will be equally affected by climate change. New regional conditions may promote new invasions or the spread of established invasive populations, especially in France and Great Britain.

A new interaction between insects and carnivorous plants is reported from Brazil. Larvae of the predatory flower fly Toxomerus basalis (Diptera: Syrphidae: Syrphinae) have been found scavenging on the sticky leaves of several carnivorous sundew species (Drosera, Droseraceae) in Minas Gerais and São Paulo states, SE Brazil. This syrphid apparently spends its whole larval stage feeding on prey trapped by Drosera leaves. The nature of this plant-animal relationship is discussed, as well as the Drosera species involved, and locations where T. basalis was observed. 180 years after the discovery of this flower fly species, its biology now has been revealed. This is (1) the first record of kleptoparasitism in the Syrphidae, (2) a new larval feeding mode for this family, and (3) the first report of a dipteran that shows a kleptoparasitic relationship with a carnivorous plant with adhesive flypaper traps. The first descriptions of the third instar larva and puparium of T. basalis based on Scanning Electron Microscope analysis are provided.

Based on an integrative taxonomic approach, we examine the differentiation of Southeast Asian snail-eating turtles using information from 1863 bp of mitochondrial DNA, 12 microsatellite loci, morphology and a correlative species distribution model. Our analyses reveal three genetically distinct groups with limited mitochondrial introgression in one group. All three groups exhibit distinct nuclear gene pools and distinct morphology. Two of these groups correspond to the previously recognized species Malayemys macrocephala (Chao Phraya Basin) and M. subtrijuga (Lower Mekong Basin). The third and genetically most divergent group from the Khorat Basin represents a previously unrecognized species, which is described herein. Although Malayemys are extensively traded and used for religious release, only few studied turtles appear to be translocated by humans. Historic fluctuations in potential distributions were assessed using species distribution models (SDMs). The Last Glacial Maximum (LGM) projection of the predictive SDMs suggests two distinct glacial distribution ranges, implying that the divergence of M. macrocephala and M. subtrijuga occurred in allopatry and was triggered by Pleistocene climate fluctuations. Only the projection derived from the global circulation model MIROC reveals a distinct third glacial distribution range for the newly discovered Malayemys species.

The quantification of realized niche overlap and the integration of species distribution models (SDMs) with calibrated phylogenies to study niche evolution are becoming not only powerful tools to understand speciation events, but can also be used as proxies regarding the delimitation of cryptic species. We applied these techniques in order to unravel how the fundamental niche evolved during cladogenesis within the Tarentola mauritanica species-complex. Our results suggest that diversification within this complex, during the Miocene and Pleistocene, is associated with both niche divergence and niche conservatism, with a pattern that varies depending on whether the variables involved are related to the mean or seasonality of temperature and humidity. Moreover, climatic variables related to humidity and temperature seasonality were involved in the niche shift and genetic diversification of the European/North African clade during the Pleistocene and in its maintenance in a fundamental niche distinct from that of the remaining members of the group. This study further highlights the need for a taxonomic revision of the T. mauritanica species-complex.

Body shape reflects species' evolution and mediates its role in the environment as it integrates gene expression, life style, and structural morphology. Its comparative analysis may reveal insight on what shapes shape, being a useful approach when other evidence is lacking. Here we investigated evolutionary patterns of body shape in the highly diverse phytophagous chafers (Scarabaeidae: Pleurosticti), a polyphagous group utilizing different parts of angiosperms. Because the reasons of their successful diversification are largely unknown, we used a phylogenetic tree and multivariate analysis on twenty linear measurements of body morphology including all major Pleurosticti lineages to infer patterns of morphospace covariation and divergence. The chafer's different feeding types resulted to be not distinguishable in the described morphospace which was largely attributed to large occupancy of the morphospace of some feeding types and to multiple convergences of feeding behavior (particularly of anthophagy). Low correlation between molecular and morphological rates of evolution, including significant rate shifts for some lineages, indicated directed selection within feeding types. This is supported by morphospace divergence within feeding types and convergent evolution in Australian Melolonthinae. Traits driving morphospace divergence were extremities and traits linked with locomotion behavior, but also body size. Being highly adaptive for burrowing and locomotion these traits showed major changes in the evolution of pleurostict scarabs. These activities also affected another trait, the metacoxal length, which is highly influenced by key innovations of the metacoxa (extended mesal process, secondary closure) particularly in one lineage, the Sericini. Significant shape divergence between major lineages and a lack of strong differentiation among closely related lineages indicated that the question about the presence or absence of competition-derived directed selection needs to be addressed for different time scales. Striking divergence between some sister lineages at their origin revealed strong driven selection towards morphospace divergence, possibly linked with resource partitioning.

We used pitfall trapping to investigate the effects of elevation, plant density and soil structure on species diversity and the impact of these habitat factors on lizard habitat selectivity in the Qom Province in the Central Plateau of Iran. From a total of 12 1-ha plots, we captured 363 individuals of 15 species of lizards (six species of Lacertidae, five species of Agamidae, two species of Gekkonidae, one species of Varanidae and one species of Scincidae). A generalized linear model (GLM) determined that elevation was the most important factor impacting species diversity. The highest species diversity was at the intermediate elevation (1289 m). Abundance of 6 out of 15 species showed strong relationships with some habitat factors. These relationships were demonstrated by habitat selectivity index (Ivlev's index). Our result supports other surveys that showed that elevation plays an important role in determining lizard species diversity.

While traditionally species recognition has been based solely on morphological differences either typological or quantitative, several newly developed methods can be used for a more objective and integrative approach on species delimitation. This may be especially relevant when dealing with cryptic species or species complexes, where high overall resemblance between species is coupled with comparatively high morphological variation within populations. Rock lizards, genus Darevskia, are such an example, as many of its members offer few diagnostic morphological features. Herein, we use a combination of genetic, morphological and ecological criteria to delimit cryptic species within two species complexes, D. chlorogaster and D. defilippii, both distributed in northern Iran. Our analyses are based on molecular information from two nuclear and two mitochondrial genes, morphological data (15 morphometric, 16 meristic and four categorical characters) and eleven newly calculated spatial environmental predictors. The phylogeny inferred for Darevskia confirmed monophyly of each species complex, with each of them comprising several highly divergent clades, especially when compared to other congeners. We identified seven candidate species within each complex, of which three and four species were supported by Bayesian species delimitation within D. chlorogaster and D. defilippii, respectively. Trained with genetically determined clades, Ecological Niche Modeling provided additional support for these cryptic species. Especially those within the D. defilippii-complex exhibit well-differentiated niches. Due to overall morphological resemblance, in a first approach PCA with mixed variables only showed the separation between the two complexes. However, MANCOVA and subsequent Discriminant Analysis performed separately for both complexes allowed for distinction of the species when sample size was large enough, namely within the D. chlorogaster-complex. In conclusion, the results support four new species, which are described herein.

The climatic cycles of the Quaternary, during which global mean annual temperatures have regularly changed by 5-10°C, provide a special opportunity for studying the rate, magnitude, and effects of geographic responses to changing climates. During the Quaternary, high- and mid-latitude species were extirpated from regions that were covered by ice or otherwise became unsuitable, persisting in refugial retreats where the environment was compatible with their tolerances. In this study we combine modern geographic range data, phylogeny, Pleistocene paleoclimatic models, and isotopic records of changes in global mean annual temperature, to produce a temporally continuous model of geographic changes in potential habitat for 59 species of North American turtles over the past 320 Ka (three full glacial-interglacial cycles). These paleophylogeographic models indicate the areas where past climates were compatible with the modern ranges of the species and serve as hypotheses for how their geographic ranges would have changed in response to Quaternary climate cycles. We test these hypotheses against physiological, genetic, taxonomic and fossil evidence, and we then use them to measure the effects of Quaternary climate cycles on species distributions. Patterns of range expansion, contraction, and fragmentation in the models are strongly congruent with (i) phylogeographic differentiation; (ii) morphological variation; (iii) physiological tolerances; and (iv) intraspecific genetic variability. Modern species with significant interspecific differentiation have geographic ranges that strongly fluctuated and repeatedly fragmented throughout the Quaternary. Modern species with low genetic diversity have geographic distributions that were highly variable and at times exceedingly small in the past. Our results reveal the potential for paleophylogeographic models to (i) reconstruct past geographic range modifications, (ii) identify geographic processes that result in genetic bottlenecks; and (iii) predict threats due to anthropogenic climate change in the future.

The amplified fragment length polymorphisms (AFLP) method has become an attractive tool in phylogenetics due to the ease with which large numbers of characters can be generated. In contrast to sequence-based phylogenetic approaches, AFLP data consist of anonymous multilocus markers. However, potential artificial amplifications or amplification failures of fragments contained in the AFLP data set will reduce AFLP reliability especially in phylogenetic inferences. In the present study, we introduce a new automated scoring approach, called "AMARE" (AFLP MAtrix REduction). The approach is based on replicates and makes marker selection dependent on marker reproducibility to control for scoring errors. To demonstrate the effectiveness of our approach we record error rate estimations, resolution scores, PCoA and stemminess calculations. As in general the true tree (i.e. the species phylogeny) is not known, we tested AMARE with empirical, already published AFLP data sets, and compared tree topologies of different AMARE generated character matrices to existing phylogenetic trees and/or other independent sources such as morphological and geographical data. It turns out that the selection of masked character matrices with highest resolution scores gave similar or even better phylogenetic results than the original AFLP data sets.

Marine invertebrates inhabiting the high Antarctic continental shelves are challenged by disturbance of the seafloor by grounded ice, low but stable water temperatures and variable food availability in response to seasonal sea-ice cover. Though a high diversity of life has successfully adapted to such conditions, it is generally agreed that during the Last Glacial Maximum (LGM) the large-scale cover of the Southern Ocean by multi-annual sea ice and the advance of the continental ice sheets across the shelf faced life with conditions, exceeding those seen today by an order of magnitude. Conditions prevailing at the LGM may have therefore acted as a bottleneck event to both the ecology as well as genetic diversity of today's fauna. Here, we use for the first time specific Species Distribution Models (SDMs) for marine arthropods of the Southern Ocean to assess effects of habitat contraction during the LGM on the three most common benthic caridean shrimp species that exhibit a strong depth zonation on the Antarctic continental shelf. While the shallow-water species Chorismus antarcticus and Notocrangon antarcticus were limited to a drastically reduced habitat during the LGM, the deep-water shrimp Nematocarcinus lanceopes found refuge in the Southern Ocean deep sea. The modeling results are in accordance with genetic diversity patterns available for C. antarcticus and N. lanceopes and support the hypothesis that habitat contraction at the LGM resulted in a loss of genetic diversity in shallow water benthos.

Background: Published nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants) are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce., Findings: Here we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis). We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist) and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers)., Conclusions: The amplified nucleotide sequences are (a) with high probability from single-copy genes, (b) easily generated at low financial costs, especially when compared to phylogenomic approaches, (c) easily sequenced by means of an additionally provided set of sequencing primers, and (d) suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

Island radiations have played a major role in shaping our current understanding of allopatric, sympatric and parapatric speciation. However, the fact that species divergence correlates with island size emphasizes the importance of geographic isolation (allopatry) in speciation. Based on molecular and morphological data, we investigated the diversification of the land snail genus Theba on the two Canary Islands of Lanzarote and Fuerteventura. Due to the geological history of both islands, this study system provides ideal conditions to investigate the interplay of biogeography, dispersal ability and differentiation in generating species diversity. Our analyses demonstrated extensive cryptic diversification of Theba on these islands, probably driven mainly by non-adaptive allopatric differentiation and secondary gene flow. In a few cases, we observed a complete absence of gene flow among sympatrically distributed forms suggesting an advanced stage of speciation. On the Jandía peninsula genome scans suggested genotype-environment associations and potentially adaptive diversification of two closely related Theba species to different ecological environments. We found support for the idea that genetic differentiation was enhanced by divergent selection in different environments. The diversification of Theba on both islands is therefore best explained by a mixture of non-adaptive and adaptive speciation, promoted by ecological and geomorphological factors.

The aim of our study was to test the robustness and efficiency of maximum likelihood with respect to different long branch effects on multiple-taxon trees. We simulated data of different alignment lengths under two different 11-taxon trees and a broad range of different branch length conditions. The data were analyzed with the true model parameters as well as with estimated and incorrect assumptions about among-site rate variation. If length differences between connected branches strongly increase, tree inference with the correct likelihood model assumptions can fail. We found that incorporating invariant sites together with Γ distributed site rates in the tree reconstruction (Γ+I) increases the robustness of maximum likelihood in comparison with models using only Γ. The results show that for some topologies and branch lengths the reconstruction success of maximum likelihood under the correct model is still low for alignments with a length of 100,000 base positions. Altogether, the high confidence that is put in maximum likelihood trees is not always justified under certain tree shapes even if alignment lengths reach 100,000 base positions.

Martialinae are pale, eyeless and probably hypogaeic predatory ants. Morphological character sets suggest a close relationship to the ant subfamily Leptanillinae. Recent analyses based on molecular sequence data suggest that Martialinae are the sister group to all extant ants. However, by comparing molecular studies and different reconstruction methods, the position of Martialinae remains ambiguous. While this sister group relationship was well supported by Bayesian partitioned analyses, Maximum Likelihood approaches could not unequivocally resolve the position of Martialinae. By re-analysing a previous published molecular data set, we show that the Maximum Likelihood approach is highly appropriate to resolve deep ant relationships, especially between Leptanillinae, Martialinae and the remaining ant subfamilies. Based on improved alignments, alignment masking, and tree reconstructions with a sufficient number of bootstrap replicates, our results strongly reject a placement of Martialinae at the first split within the ant tree of life. Instead, we suggest that Leptanillinae are a sister group to all other extant ant subfamilies, whereas Martialinae branch off as a second lineage. This assumption is backed by approximately unbiased (AU) tests, additional Bayesian analyses and split networks. Our results demonstrate clear effects of improved alignment approaches, alignment masking and data partitioning. We hope that our study illustrates the importance of thorough, comprehensible phylogenetic analyses using the example of ant relationships.

Background: Several felids are endangered and threatened by the illegal wildlife trade. Establishing geographic origin of tissues of endangered species is thus crucial for wildlife crime investigations and effective conservation strategies. As shown in other species, stable isotope analysis of hydrogen and oxygen in hair (δD(h), δ(18)O(h)) can be used as a tool for provenance determination. However, reliably predicting the spatial distribution of δD(h) and δ(18)O(h) requires confirmation from animal tissues of known origin and a detailed understanding of the isotopic routing of dietary nutrients into felid hair., Methodology/findings: We used coupled δD(h) and δ(18)O(h) measurements from the North American bobcat (Lynx rufus) and puma (Puma concolor) with precipitation-based assignment isoscapes to test the feasibility of isotopic geo-location of felidae. Hairs of felid and rabbit museum specimens from 75 sites across the United States and Canada were analyzed. Bobcat and puma lacked a significant correlation between H/O isotopes in hair and local waters, and also exhibited an isotopic decoupling of δ(18)O(h) and δD(h). Conversely, strong δD and δ(18)O coupling was found for key prey, eastern cottontail rabbit (Sylvilagus floridanus; hair) and white-tailed deer (Odocoileus virginianus; collagen, bone phosphate)., Conclusions/significance: Puma and bobcat hairs do not adhere to expected pattern of H and O isotopic variation predicted by precipitation isoscapes for North America. Thus, using bulk hair, felids cannot be placed on δ(18)O and δD isoscapes for use in forensic investigations. The effective application of isotopes to trace the provenance of feline carnivores is likely compromised by major controls of their diet, physiology and metabolism on hair δ(18)O and δD related to body water budgets. Controlled feeding experiments, combined with single amino acid isotope analysis of diets and hair, are needed to reveal mechanisms and physiological traits explaining why felid hair does not follow isotopic patterns demonstrated in many other taxa.