Enigmatic ear stones: what we know about the functional role and evolution of fish otoliths

Otoliths in bony fishes play an important role in the senses of balance and hearing. Otolith mass and shape are, among others, likely to be decisive factors influencing otolith motion and thus ear functioning. Yet our knowledge of how exactly these factors influence otolith motion is incomplete. In addition, experimental studies directly investigating the function of otoliths in the inner ear are scarce and yield partly conflicting results. Herein, we discuss questions and hypotheses on how otolith mass and shape, and the relationship between the sensory epithelium and overlying otolith, influence otolith motion. We discuss (i) the state‐of‐the‐art knowledge regarding otolith function, (ii) gaps in knowledge that remain to be filled, and (iii) future approaches that may improve our understanding of the role of otoliths in ear functioning. We further link these functional questions to the evolution of solid teleost otoliths instead of numerous tiny otoconia as found in most other vertebrates. Until now, the selective forces and/or constraints driving the evolution of solid calcareous otoliths and their diversity in shape in teleosts are largely unknown. Based on a data set on the structure of otoliths and otoconia in more than 160 species covering the main vertebrate groups, we present a hypothetical framework for teleost otolith evolution. We suggest that the advent of solid otoliths may have initially been a selectively neutral ‘by‐product’ of other key innovations during teleost evolution. The teleost‐specific genome duplication event may have paved the way for diversification in otolith shape. Otolith shapes may have evolved along with the considerable diversity of, and improvements in, auditory abilities in teleost fishes. However, phenotypic plasticity may also play an important role in the creation of different otolith types, and different portions of the otolith may show different degrees of phenotypic plasticity. Future studies should thus adopt a phylogenetic perspective and apply comparative and methodologically integrative approaches, including fossil otoliths, when investigating otoconia/otolith evolution and their function in the inner ear.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Thermal habitat segregation among morphotypes of whitefish (Coregonus lavaretus: Salmonidae) and invasive vendace (C. albula): a mechanism for co‐existence?

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Geophilomorph centipedes from the Cretaceous amber of Burma

The only previously known Mesozoic fossils of the chilopod order Geophilomorpha are two species from the Late Jurassic and Late Cretaceous, both known from single specimens that cannot be assigned with precision to a family. Four specimens from the Late Cretaceous (earliest Cenomanian) amber of Burma include three that can be identified as conspecific, described here as Kachinophilus pereirai gen. nov. sp. nov. These specimens preserve greater morphological detail in comparison with other fossil geophilomorphs: the form and fine features of the head, the maxillary complex, the trunk sternites with associated glandular pores and the ultimate pair of legs defend the assignment of the species to the extant family Geophilidae, and most probably to a derived subgroup including well‐known extant genera such as Ribautia Brölemann, 1909. Confocal laser scanning microscopy supplements examination under incident and transmitted light to document details of high taxonomic relevance in the head and the forcipular segment. The modern appearance of this species and its membership among deeply nested extant clades are consistent with molecular estimates that most of the diversity of crown‐group Geophilomorpha originated before the Late Cretaceous.     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.     

Saccular otolith mass asymmetry in adult flatfishes

A dimensionless measure of otolith mass asymmetry, χ, was calculated as the difference between the masses of the right and left paired otoliths divided by average otolith mass. Saccular otolith mass asymmetry was studied in eight flatfish species (110 otolith pairs) and compared with data from a previously published study on roundfishes. As in the case of symmetrical fishes, the absolute value of χin flatfishes does not depend on fish length and otolith growth rate, although otolith mass and the absolute value of otolith mass difference are correlated with fish length. The values of χwere between ?0·2 and +0·2 in 96·4% of flatfishes studied. The mean ±s .e . value of χin flatfishes was significantly larger than in standard bilaterally symmetrical marine fishes (‘roundfishes’), respectively 0·070 ± 0·006 and 0·040 ± 0·006. The most prominent distinction is the existence of downside prevalence of saccular otolith mass in flatfishes, which contrasts with no right–left prevalence in roundfishes found in a previous study. In the right‐eyed flatfishes (Soleidae), the left saccular otoliths are heavier than the right otoliths. In the left‐eyed flatfishes (Bothidae and Citharidae), the right saccular otoliths are heavier than the left otoliths. Not all flatfishes, however, fit in this design: 11·8% of flatfishes studied had the heavier saccular otoliths in the upside labyrinth and 5·4% of flatfishes had no otolith mass asymmetry (within the accuracy of the analysis). At the same time, the more mobile flatfishes (bothids and citharids) have more symmetrical and, hence, more precisely organized saccular otolith organs than the bottom‐associated flatfishes (soleids). It is possible to assume that the value of the otolith asymmetry is not only correlated with flatfish placement in a particular family, or position of eyes, but also may correlate with general aspects of their ecology. Mathematical modelling indicated that for most flatfishes one‐side saccular prevalence had no substantial significance for sound processing. On the other hand, calculations showed that 49% of flatfishes (but only 14·5% of roundfishes) have |χ| which exceed the critical level and, in principle, could sense the difference between the static displacement of the large and small paired otoliths. At that, the number of the soleids that could sense this difference is greater than the number of the bothids and citharids, 84 and 27%, respectively.     

Re‐evaluation of the ontogeny and reproductive biology of the Triassic fish Saurichthys (Actinopterygii,Saurichthyidae)

Viviparity has evolved independently at least 12 times in ray‐finned fishes. However, the fossil record of actinopterygian viviparity is poor, with only two documented occurrences. Both of these are from the non‐teleost actinopterygian Saurichthys, and include S. curionii and S. macrocephalus from the Middle Triassic Meride Limestone (Monte San Giorgio, Switzerland). Here, we present new data on the reproductive biology of these species, giving unprecedented insights into their life‐history. Based on positional and preservational criteria, six specimens were identified as unambiguously gravid. Embryos were positioned dorsal to the gastrointestinal tract, parallel to the axial skeleton and to each other, in the posterior two‐thirds of the abdominal region. A minimum of 16 embryos are preserved in the most fecund females and, based on the largest preserved embryos and smallest preserved neonates, birth must have occurred at 7–12% of maternal fork length. Embryonic crania and teeth are relatively well‐ossified, however ossification of the parietal region is delayed. In the postcranium, the median scale rows and lepidotrichia are ossified, but not the lateral scale rows. Ossified squamation and gradual allometric growth suggests that neonates did not undergo metamorphosis and were relatively precocial. When considered in a phylogenetic context, neither live birth nor internal fertilization appears to represent the primitive state for saurichthyid fishes.     

Dietary histories of herbivorous loricariid catfishes: evidence from δ<Superscript>13</Superscript>C values of otoliths

The ecology of many Neotropical fishes is difficult or often impossible to study during rainy seasons. Thus, ecological studies of tropical fishes are usually performed on fish captured only during dry seasons. Because otoliths preserve a record of life history, this study evaluated the utility of otolith stable isotope values for the investigation of trophic ecology of Neotropical fishes (specifically herbivorous loricariid catfish) throughout their lives. Because plant dietary materials have δ¹³C values that are determined by their photosynthetic pathways, metabolism and environmental conditions, different plants may impart different isotope values on fish otoliths that reflect consumption of these plants. The δ¹³C_(otolith) values of xylophagous Panaque nigrolineatus captured in the field were significantly lower than those of algivorous Hypostomus regani from a nearby region. A laboratory experiment wherein Hypostomus sp. had δ¹³C_(otolith) values that reflected the δ¹³C values of their plant diet and additional evidence indicate that δ¹³C_(otolith) values in loricariid catfish otoliths can record dietary history.     

A new phylogenetic hypothesis of turtles with implications for the timing and number of evolutionary transitions to marine lifestyles in the group

Evolutionary transitions to marine habitats occurred frequently among Mesozoic reptiles. Only one such clade survives to the present: sea turtles (Chelonioidea). Other marine turtles originated during the Mesozoic, but uncertain affinities of key fossils have obscured the number of transitions to marine life, and the timing of the origin of marine adaptation in chelonioids. Phylogenetic studies support either a highly‐inclusive chelonioid total‐group including fossil marine clades from the Jurassic and Cretaceous (e.g. protostegids, thalassochelydians, sandownids) or a less inclusive chelonioid total‐group excluding those clades. Under this paradigm, these clades belong outside Cryptodira, and represent at least one additional evolutionary transition to marine life in turtles. We present a new phylogenetic hypothesis informed by high resolution computed tomographic data of living and fossil taxa. Besides a well‐supported Chelonioidea, which includes protostegids, we recover a previously unknown clade of stem‐group turtles, Angolachelonia, which includes the Late Jurassic thalassochelydians, and the Cretaceous–Palaeogene sandownids. Accounting for the Triassic Odontochelys, our results indicate three independent evolutionary transitions to marine life in non‐pleurodiran turtles (plus an additional two‐three in pleurodires). Among all independent origins of marine habits, a pelagic ecology only evolved once, among chelonioids. All turtle groups that independently invaded marine habitats in the Jurassic–Cretaceous (chelonioids, angolachelonians, bothremydid pleurodires) survived the Cretaceous–Palaeogene mass extinction event. This highlights extensive survival of marine turtles compared to other marine reptiles. Furthermore, deeply‐nested clades such as chelonioids are found by the middle Early Cretaceous, suggesting a rapid diversification of crown‐group turtles during the Early Cretaceous.     

A new commelinid monocot seed fossil from the early Eocene previously identified as Solanaceae

Premise of the Study

Fossils provide minimum age estimates for extant lineages. Here we critically evaluate Cantisolanum daturoides Reid & Chandler and two other early putative seed fossils of Solanaceae, an economically important plant family in the Asteridae.

Methods

Three earliest seed fossil taxa of Solanaceae from the London Clay Formation (Cantisolanum daturoides) and the Poole and Branksome Sand Formations (Solanum arnense Chandler and Solanispermum reniforme Chandler) were studied using x‐ray microcomputed tomography (MCT) and scanning electron microscopy (SEM).

Key Results

The MCT scans of Cantisolanum daturoides revealed a high level of pyrite preservation at the cellular level. Cantisolanum daturoides can be clearly excluded from Solanaceae and has more affinities to the commelinid monocots based on a straight longitudinal axis, a prominent single layer of relatively thin‐walled cells in the testa, and a clearly differentiated micropyle surrounded by radially elongated and inwardly curved testal cells. While the MCT scans show no internal preservation in Solanum arnense and Solanispermum reniforme, SEM images show the presence of several characteristics that allow the placement of these taxa at the stem node of Solanaceae.

Conclusions

Cantisolanum daturoides is likely a member of commelinid monocots and not Solanaceae as previously suggested. The earliest fossil record of Solanaceae is revised to consist of fruit fossil with inflated calyces from the early Eocene of Patagonia (52 Ma) and fossilized seeds from the early to mid‐Eocene of Europe (48–46 Ma). The new identity for Cantisolanum daturoides does not alter a late Cretaceous minimum age for commelinids.     

Development of the pancreas in medaka,Oryzias latipes,from embryo to adult

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1‐ and ptf1a‐transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.     

Potential biological control of Erwinia tracheiphila by internal alimentary canal interactions in Acalymma vittatum with Pseudomonas fluorescens

Aims

We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum.

Methods and Results

Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal.

Conclusions

Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient‐limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co‐occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector.

Significance and Impact of the Study

This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.     

Bioassay‐Guided Isolation of Antifungal Compounds from Disporopsis aspersa (Hua) Engl. ex Diels against Pseudoperonospora cubensis and Phytophthora infestans

Oomycetes are one type of the most highly destructive of the diseases that cause damage to some important crop plants, such as potato late blight, cucumber downy mildew, and grape downy mildew. As main approach of the ongoing search for new botanical fungicide from plant, the secondary metabolites of D. aspersa were investigated. Through efficient bioassay‐guided isolation, two new ( 1 and 2 ) and 12 known compounds ( 3  –  14 ) were isolated, and their structures were determined via extensive NMR, HR‐ESI‐MS, and IR. They were isolated from this genus for the first time except for compounds 11 and 12 . The biological properties of 1  –  14 were evaluated against Pseudoperonospora cubensis and Phytophthora infestans. Compounds 1  –  8 showed potent antifungal activity in vitro. Additionally, compound 3 has preferable control effect on cucumber downy mildew, showing dual effect of protection and treatment in vivo.     

Otoliths of Five Extant Species of the Annual Killifish Nothobranchius from the East African Savannah

This study presents, for the first time, a comprehensive dataset that documents the range of inter- and intraspecific otolith variation in aplocheiloid killifish, based on a total of 86 individuals representing five extant species of Nothobranchius PETERS, 1868, from East Africa: the sympatric pairs N. rubripinnis SEEGERS, 1986 and N. ruudwildekampi COSTA, 2009 (Eastern Tanzania), and N. orthonotus (PETERS, 1844) and N. furzeri JUBB, 1971 (Southern Mozambique), and two isolated populations of N. korthausae MEINKEN, 1973 (Eastern Tanzania). Otolith characters were analysed based on SEM images, and otolith morphometry was conducted using uni- and multivariate statistics. Two ancient clades of probably Early to Middle Miocene age in eastern Tanzania and southern Mozambique can be recognized based on otolith morphologies, which is consistent with previous work based on molecular data. The distinctive sulcus morphologies in the otoliths of sympatric species may be linked to species-specific hearing capabilities, perhaps constituting a case of character displacement in an area of secondary sympatry. The otoliths of the studied species of Nothobranchius are diagnostic at the species level, even in the case of closely related species diagnosable otherwise only by minor differences in coloration. The two populations of N. korthausae also displayed some differences in their otolith characters. The new data may facilitate future recognition of fossil species of Nothobranchius. As no fossil remains of extant aplocheiloid killifishes have yet been described, the discovery of fossil otoliths of Nothobranchius would significantly advance understanding of the evolutionary history of this interesting group of fishes.     

Teruelia diezii gen. et sp. nov.: an early polysporangiophyte from the Lower Devonian of the Iberian Peninsula

A new basal land plant, Teruelia diezii gen. et sp. nov., is described from the shallow‐water marine deposits of the Lower Devonian (Lochkovian–Pragian) Nogueras Formation of the Iberian Peninsula (north Gondwana palaeocontinent). Teruelia is preserved as a compression fossil and consists of isotomously branched, robust stems terminated in large, fusiform, twisted sporangia. This morphology suggests that Teruelia is very probably equivalent to Aglaophyton, a permineralized early polysporangiophyte known up to now only from the Lower Devonian (early Pragian to ?earliest Emsian) Rhynie Chert in Scotland (Laurussia palaeocontinent), which represents an early terrestrial hot‐spring ecosystem. Accepted phylogenies identify Aglaophyton as sister to vascular plants. Our phylogeny‐based results identify the Aglaophyton/Teruelia biological entity (i.e. Aglaophyton anatomical characters plus Teruelia external morphology) as the most direct vascular plant precursor. It shows that at least one Rhynie Chert type plant had a much wider distribution than previously known and suggests that Aglaophyton was not restricted to hydrothermal environments, unlike other Rhynie Chert plants.     

Orchid seed removal by ants in Neotropical ant‐gardens

• Most plants that inhabit ant‐gardens (AGs) are cultivated by the ants. Some orchids occur in AGs; however, it is not known whether their seeds are dispersed by AG ants because most orchid seeds are tiny and dispersed by wind.
• We performed in situ seed removal experiments, in which we simultaneously provided Azteca gnava ants with seeds of three AG orchid species and three other AG epiphyte species (Bromeliaceae, Cactaceae and Gesneriaceae), as well as the non‐AG orchid Catasetum integerrimum.
• The seeds most removed were those of the bromeliad Aechmea tillandsioides and the gesneriad Codonanthe uleana, while seeds of AG orchids Coryanthes picturata, Epidendrum flexuosum and Epidendrum pachyrachis were less removed. The non‐AG orchid was not removed. Removal values were positively correlated with the frequency of the AG epiphytes in the AGs, and seeds of AG orchids were larger than those of non‐AG orchids, which should favour myrmecochory.
• Our data show that Azt. gnava ants discriminate and preferentially remove seeds of the AG epiphytes. We report for the first time the removal of AG orchid seeds by AG ants in Neotropical AGs.

     

Evolutionary significance of a middle Cambrian (Series 3) in situ occurrence of the pedunculate rhynchonelliform brachiopod Nisusia sulcata

Exceptionally preserved, silicified and articulated complete shells of the rhynchonelliform kutorginate brachiopod Nisusia sulcata are redescribed from the middle Cambrian (Series 3) Marjum Limestone, Utah. Cylindroid sausage‐like protrusions, emerging posteriorly between the valves, were originally interpreted as faecal in origin, but restudy under the SEM shows that these features represent silicified pedicles as they are attached in situ to other Nisusia. The Nisusia host most likely was alive at the time of attachment. Restudy of the pedicles of Nisusia provides new phylogenetic information on the anatomy of the earliest rhynchonelliforms. The silicified pedicles differ considerably from the pedicles of living crown group rhynchonelliforms in being strongly annulated, distally tapering, and were likely to have been rather more flexible. The Nisusia pedicles are more similar to the exceptionally preserved pedicles from other Cambrian rhynchonelliform brachiopods, including Kutorgina, Longtancunella and Alisina, but these emerge from the ventral apical foramen rather than from between the valves as in Nisusia. Although generally similar, these two types of pedicles are unlikely to represent homologous structures as Nisusia is provided with both an apical foramen (possibly larval attachment) and a posterior adult pedicle. The similarities may be explained by similar type of accretionary growth from two different types of epithelia. The Nisusia‐like pedicle appeared early within the kutorginates and rhynchonellates. The discovery of hollow spines in Nisusia sulcata further supports the generic assignation of the species.     

Changes in lifestyle and habitat of Zoophycos‐producing animals related to evolution of phytoplankton during the Late Mesozoic: geological evidence for the ‘benthic‐pelagic coupling model’

The trace fossil Zoophycos characterized by complex, three‐dimensional morphology with systematic internal structures occurs throughout the Phanerozoic marine sediments. The specimens of Zoophycos examined herein consist of a downward and helical spreite and are a product of the excretory behaviour of endobenthic detritus feeders. They are divided into two basic types: pre‐Jurassic and post‐Cretaceous types on the basis of whorls of spreiten in a single specimen. The pre‐Jurassic type has fewer than four whorls. In contrast, most of the post‐Cretaceous specimens exhibit spreite with multiple coils more than ten whorls. The abrupt increase in whorl number during the Cretaceous suggests that the sedentary lifestyle of the producer should change from a short‐term stay to long‐term or permanent occupation of the same burrow. Timing of the lifestyle change the Zoophycos producers seems to be closely related to the deep‐seaward migration of their habitats. The change in lifestyle and migration of Zoophycos‐producing animals during the Cretaceous might be attributable to the establishment of eutrophic bottom conditions in the deep sea. These changes seem to be associated with the flux of large amounts of phytodetrital food produced by phytoplankton, which experienced an explosive increase in species diversity during the Late Jurassic to the Late Cretaceous. The series of changes in lifestyle and habitat of the Zoophycos animals during the Late Mesozoic can serve as one piece of geological evidence for the ‘benthic‐pelagic coupling model’.     

Drought,post‐dispersal seed predation,and the establishment of epiphytic bromeliads (Tillandsia spp.)

Post‐dispersal, epiphyte seed predation is poorly documented. Our study on the in situ germination of two Tillandsia species in a deciduous forest in Yucatan, Mexico showed an average post‐dispersal seed predation rate by the Yucatan deer mouse (Peromyscus yucatanicus) of 90%. Post‐dispersal predation was thus more limiting than drought.