Cytogenetic analyses of eight species in the genus <Emphasis Type="Italic">Leptodactylus</Emphasis> Fitzinger, 1843 (Amphibia,Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations

Background

The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation.

Results

Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents.

Conclusions

Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.

     

A New Skull of <Emphasis Type="Italic">Hyaenictis</Emphasis> Gaudry, 1861 (Carnivora,Hyaenidae) Shows Incipient Adaptations to Durophagy

The European Miocene records a wide diversity of hyaenid ecomorphotypes represented by multiple genera. Among these, Hyaenictis Gaudry, 1861, is one of the least known. This genus includes four species from the late Miocene and Pliocene of the Old World, but in Europe Hyaenictis is only represented by two species, recorded by scarce and fragmentary remains: Hyaenictis graeca Gaudry, 1861, from Pikermi (MN12; Greece) and Hyaenictis almerai Villalta Comella and Crusafont Pairó, 1948, from Sant Miquel de Toudell (MN10; Vallès-Penedès Basin, NE Iberia). Here, we describe a new skull of Hyaenictis aff. almerai from the Vallès-Penedès site of Ronda Oest Sabadell Sector D (MN10), representing the most complete European specimen of the genus. In the presence of m2 and virtual lack of m1 metaconid, the described cranium more closely resembles Hyaenictis rather than any other medium- to large-sized European hyaenid. However, the new skull does not fit well with previously known Hyaenictis species, more closely resembling the bone-cracking Adcrocuta Kretzoi, 1938, in the development of premolar accessory cuspids and the possession of relatively broad cheek teeth. These and other features (strong mandibular muscular insertions and enamel microstructure) denote more durophagous adaptations than previously documented in Hyaenictis (considered a cursorial/dog-like hyaena), and favor the inclusion of H. aff. almerai in the transitional bone-cracking hyaenid ecomorphotype.     

Two new species of <Emphasis Type="Italic">Arachnothryx</Emphasis> (Rubiaceae) from Oaxaca,Mexico

Two new species of Arachnothryx (Guettardeae, Rubiaceae) from the cloud forest of the Sierra Norte of Oaxaca, Mexico are described and illustrated. Arachnothryx axillaris together with A. heteranthera are the only two species in the genus with axillary inflorescences, but otherwise they are not similar. The new species is more similar to A. capitellata, but it differs in pubescence and inflorescence morphology. Arachnothryx flores-olverae can be confused with A. buddleioides mainly because they share discolorous leaves with densely tomentose pubescence, but they differ in stipule and inflorescence morphology. With these two new species the Sierra Norte of Oaxaca becomes more evidently the main hotspot of species diversity for the genus.     

Letting the Air Out: <Emphasis Type="Italic">Aire</Emphasis> as an Empty Signifier in Oaxacan Understandings of Illness

“Air (aire, also aigre) in the body” is a frequent explanation of illness according to the traditional medical beliefs in Mexico. Anthropologists have generally scrutinized aire in the context of other common folk illnesses treated by traditional healers (curanderas). However, drawing on my research in the communities of Northern Oaxaca I suggest that aire occupies a more distinct position in the folk medical cosmology than it has usually been credited with. This distinction rests on the notion’s exceptional ambivalence and openness to multiple interpretations. “Air” is recurred to as the cause of illness mainly in situations where every other explanation, either “traditional” or “biomedical,” seems to be inadequate. The physical properties of air—its transparency, invisibility, apparent immateriality, near omnipresence, and virtual “nothingness”—render it a suitable explanation of the last resort. Local understandings of what aire “is” are often vague and elusive, and in many respects the term functions in folk medical discourse as an “empty signifier.”     

The genera Amblostoma,Lanium, and Stenoglossum (Orchidaceae)

The generaAmblostoma, Lanium, andStenoglossum are discussed, and it is concluded that all three should be included inEpidendrum.Lanium is treated as a section, and new names are proposed for two species whose epithets are preoccupied inEpidendrum: E. macrum (forAmblostoma gracile) andE. stiliferum (forLanium subulatum).     

New data on the distribution and host plants of weevils (Coleoptera,Curculionoidea: Apionidae,Curculionidae) in the south of Baikal Siberia and in Mongolia

Two weevil species, Rhinoncus autumnalis Korotyaev, 1980 and Orchestes medvedevi (Korotyaev, 1995), comb. n. (from Rhynchaenus), both described from Mongolia, are recorded from Russia (Buryatia) for the first time. A key to five Orchestes species associated with elms in Eastern Siberia, the Russian Far East, and Mongolia is provided. Aizobius sedi (Germar, 1818) is recorded for the first time from Eastern Siberia based on the recent findings in Buryatia and on older collections from Tuva where it is associated with Orostachys spinosa (L.) C.A. Mey. (Crassulaceae); it is also recorded for the first time from Kyrgyzstan and Mongolia. Magdalis (Aika) margaritae Barrios, 1984, native of Mongolia, Northern China and the south of the Russian Far East, is recorded from Buryatia. Lixus (Broconius) korotyaevi Ter-Minassian, 1989, formerly known in Russia only from southern Tuva, is recorded from Buryatia where it was collected on Suaeda ?prostrata Pallas, which is the first known host of this species. Rhamphus ?oxyacanthae (Marsham, 1802) is reported from Buryatia where it was collected from Cotoneaster melanocarpa, and from Mongolia.     

A novel set of 223 EST-SSR markers in <Emphasis Type="Italic">Casuarina</Emphasis> L. ex Adans.: polymorphisms,cross-species transferability,and utility for commercial clone genotyping

Simple sequence repeat (SSR) markers are very useful for genetic applications in plants, but SSR resource for the important tree genus Casuarina L. ex Adans. is still limited. In this study, we report a novel set of 223 SSR markers in Casuarina developed from expressed sequence tag (EST) resource of GenBank. The 223 EST-SSR markers were polymorphic among 10 unrelated individuals of C. equisetifolia L. Johnson, with the number of alleles per locus (N_a), observed heterozygosity (H_o), expected heterozygosity (H_e), and polymorphic information content (PIC) averaging at 5.5, 0.72, 0.86, and 0.63, respectively. The rates of cross-species transferability ranged from 96.9% (C. glauca Sieber ex Sprengel) through 97.8% (C. cunninghamiana Miquel) to 99.1% (C. junghuhniana Miquel). Fifty-five C. equisetifolia clones widely planted in China were successfully genotyped with a subset of 20 EST-SSRs. These newly developed markers will have a great potential for genetic and breeding applications in Casuarina species and related taxa.     

Revision of the taxonomic position of the Olkhon mountain vole (Rodentia,Cricetidae)

The phylogenetic position of the Olkhon mountain vole (Alticola olchonensis Litvinov, 1960) was studied using the sequences of four nuclear (BRCA, GHR, LCAT, and IRBP) and one mitochondrial (cyt. b) gene. Until now multiple studies of the systematic position of this vole had been based exclusively on morphological data, while the major taxonomic references contain contradictory information regarding both the subgeneric and species status of this animal. It was established that the molecular data and morphology data allow us to concern the Lake Baikal vole unambiguously as a part of the nominative subgenus Alticola instead of Aschizomys.     

Frequently asked questions about chlorophyll fluorescence,the sequel

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F _V /F _M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.     

Nutritional symbionts of a putative vector, <Emphasis Type="Italic">Xyleborus bispinatus</Emphasis>, of the laurel wilt pathogen of avocado, <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.     

Trilobites of the Galgenberg Member (Tannenknock Formation), middle Cambrian Stage 5, Franconian Forest,Germany: a paradigmatic lowermost middle Cambrian West Gondwanan fauna

Earliest middle Cambrian rocks in the Franconian Forest, formerly known as the ‘Galgenberg Formation’, include a moderately diverse fauna with a characteristic West Gondwanan, Atlas-type trilobite assemblage with often surprisingly well-preserved specimens. The hitherto inadequately characterised and poorly described assemblage includes Kingaspidoides frankenwaldensis, K. sp. aff. usitata, K. alberti sp. nov., K. meieri sp. nov., K.? sp. A, Ornamentaspis cf. crassilimbata, Latikingaspis sp. aff. alatus, Enixus sp. aff. juvenis, Acadoparadoxides sp. A, Parasolenopleura wurmi sp. nov., Parasolenopleura parabolica sp. nov. and Acanthomicmacca franconica Geyer, 2016. In addition to precise documentation of the species’ morphology and ontogenetic development, this study exemplifies allometric developments during the ontogeny of ellipsocephaloid and early solenopleurid trilobites, particularly Kingaspidoides and Parasolenopleura, and effects of deformation and distortion caused by diagenesis and tectonics. It further discusses the aspects of the trilobites’ ecology and taphonomy, and it characterises generic differences within the Kingaspis clade, particularly of Kingaspidoides, Latikingaspis and Ornamentaspis.     

New data on the taxonomy,distribution, and ecology of the weevil genus <Emphasis Type="Italic">Otiorhynchus</Emphasis> germar (Coleoptera,Curculionidae)

The subgenus Pocusogetus Rtt. of the genus Otiorhynchus Germ. is revised. The subgenus includes O. rosti Strl., O. shapovalovi Davidian et Yunakov, O. obsulcatus Strl., O. fischtensis Rtt., and O. gusakovi sp. n. closely related to O. fischtensis (both from Mt. Fisht, the Western Caucasus). O. fischtensis is transferred from the subgenus Vicoranius Rtt., its lectotype is designated. A key to species of Pocusogetus is given. The systematic position of the subgenera Pocusogetus and Vicoranius in the genus Otiorhynchus is discussed. New data on the geographical distribution and ecology of the little-known species of the subgenera Obvoderus Rtt., Pseudoprovadilus Magnano, and Clypeorhynchus Yunakov et Arzanov are given. Some features of ecological differentiation between Otiorhynchus species in the alpine and subalpine zones of the Caucasus are discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

New species of the buprestid genus <Emphasis Type="Italic">Endelus</Emphasis> Deyrolle (Coleoptera,Buprestidae) from China,India, and Laos

Endelus (Kubaniellus) indicus sp. n. from India, E. (K.) lao sp. n. and E. (K.) khnzoriani sp. n. from Laos, E. (s. str.) sausai sp. n. from China, and E. (s. str.) dembickyi sp. n. from India are described, the two latter species are included in the Endelus bicarinatus Théry, 1932 species-group recently established by the author. E. collinus Obenberger, 1922 is included in this group; lectotype of this species is designated. Keys to species of the subgenus Kubaniellus and of the E. collinus group are provided. E. (K.) kareni Kalashian is for the first time recorded for Shaanxi Prov., E. pacholatkoi Kalashian, E. smaragdinus Desc. et Vill., and E collinus Obenb., for Laos (the latter species, also for Myanmar).     

A review of the genera <Emphasis Type="Italic">Calotelea,Calliscelio</Emphasis>, and <Emphasis Type="Italic">Oxyscelio</Emphasis> (Scelioninae,Scelionidae, Proctotrupoidea) from the Palaearctic fauna

Nine new species of scelionids collected in Japan, Israel, and the Ukraine, Calotelea shimurai Kononova et Fursov, C. japonica Kononova, C. stellae Kononova, Calliscelio recens Kononova, C. floridum Kononova, C. parilis Kononova, C. ordo Kononova, Oxyscelio florum Kononova, and O. perpensum Kononova, are described. A brief morphological characteristics of the mentioned genera and some data on the geographical distribution of these species are given. Calotelea shimurai differs from C. striola Kononova in the sculpture of the metasoma (tergites I–III striate, whereas in C. striola striation present only on tergites I and II), fore-wing venation (stigmal vein of C. shimurai 0.43 times as long as postmarginal vein and 1.75 times as long as marginal one; in C. striola, stigmal vein 0.52 times as long as postmarginal vein and 1.3 times as long as marginal one), and the length of the metasoma (in C. shimurai and C. striola, metasoma 4.0 and 2.3 times as long as wide, respectively). Calotelea shimurai parasitizes in eggs of the dragonflies Aeshna nigroflava Martin, Planaeschna milnei Selys, and Boyeria macachlani (Aeshnidae, Odonata). C. japonica is closely related to C. originalis Kozlov and Kononova, but differs from it in the sculpture of the metasoma (metasomal tergites with longitudinal wrinkles against the bright smooth background; in C. originalis, tergites I and II with longitudinal wrinkles against the alveolate background), in the coloration of fore wing (infuscate in C. japonica and dark, with dark transverse stripes in C. originalis). C. stellae differs from C. artus Kozlov and Kononova in the more flattened mesoscutum (C. artus with protruding mesoscutum) and the sculpture of the metasomal tergites (in C. stellae, only petiolus and tergite II striate, while in C. artus, such striation present on tergites I–IV). C. stellae was reared from eggs of unidentified Orthoptera. C. recens is closely related to C. parilis Kononova. It can be distinguished by the fore-wing venation (C. recens has stigmal vein, which is twice as long as marginal vein and 0.66 times as long as postmarginal one; stigmal vein of C. parilis is 3 times as long as marginal vein and 0.83 times as long as postmarginal one), by the sculpture of the metasoma, and coloration of the coxae (yellow in C. recens and black in C. parilis). C. floridum is similar to C. mediterranea Kieffer, but can be identified by the length of the postmarginal vein, which is 3 times as long as the stigmal vein, whereas C. mediterranea has the postmarginal vein, which is only twice as long as the stigmal one. C. floridum also differs in the sculpture of the metasoma (C. floridum has all the metasomal tergites with longitudinal lines, while C. mediterranea has only metasomal petiolus with the same sculpture and tergites II–IV with alveolate sculpture, tergites V and VI are slightly stippled) and in the coloration of the legs, which are yellow (as coxae), while C. mediterranea has brownish black legs. C. parilis resembles C. recens, but differs from it in the fore-wing venation, sculpture of the metasoma, and coloration of the coxae. C. ordo differs from the closely related C. ruficollis Kozlov et Kononova in the head sculpture, which is finely alveolate in C. ordo and finely granulate in C. ruficollis. Oxyscelio florum is closely related to O. perpensum, but differs from it in the coloration of the body and size of the antennal segments, stigmal and postmarginal veins, and metasomal tergites. O. perpensum is closely related to O. florum. The main distinguishing morphological characters are similar to those in O. florum. O. perpensum was reared from eggs of unidentified Orthoptera.     

Variability,stability, and resilience of fecal microbiota in dairy cows fed whole crop corn silage

The microbiota of whole crop corn silage and feces of silage-fed dairy cows were examined. A total of 18 dairy cow feces were collected from six farms in Japan and China, and high-throughput Illumina sequencing of the V4 hypervariable region of 16S rRNA genes was performed. Lactobacillaceae were dominant in all silages, followed by Acetobacteraceae, Bacillaceae, and Enterobacteriaceae. In feces, the predominant families were Ruminococcaceae, Bacteroidaceae, Clostridiaceae, Lachnospiraceae, Rikenellaceae, and Paraprevotellaceae. Therefore, Lactobacillaceae of corn silage appeared to be eliminated in the gastrointestinal tract. Although fecal microbiota composition was similar in most samples, relative abundances of several families, such as Ruminococcaceae, Christensenellaceae, Turicibacteraceae, and Succinivibrionaceae, varied between farms and countries. In addition to the geographical location, differences in feeding management between total mixed ration feeding and separate feeding appeared to be involved in the variations. Moreover, a cow-to-cow variation for concentrate-associated families was demonstrated at the same farm; two cows showed high abundance of Succinivibrionaceae and Prevotellaceae, whereas another had a high abundance of Porphyromonadaceae. There was a negative correlation between forage-associated Ruminococcaceae and concentrate-associated Succinivibrionaceae and Prevotellaceae in 18 feces samples. Succinivibrionaceae, Prevotellaceae, p-2534-18B5, and Spirochaetaceae were regarded as highly variable taxa in this study. These findings help to improve our understanding of variation and similarity of the fecal microbiota of dairy cows with regard to individuals, farms, and countries. Microbiota of naturally fermented corn silage had no influence on the fecal microbiota of dairy cows.     

Malcofaunal Diversity of Chilika Lake,Odisha, India

Investigation during the period of 3 years from 2007 to 2010 on the malacofauna of Chilika lake revealed the occurrence of 126 molluscan taxa belonging to 56 families, 18 orders of three classes in the bottom sediment. Of these 61 species belonged to Bivalvia, 64 species belonged to Gastropoda and one species belonged to Polyplacophora. Maximum Bivalvia and Gastropoda taxa were found in the outer channel region of the lake. The dominating species were Crassostrea cuttackensis, Saccostrea cucullata, Brachidontes undulatus, Meretrix meretrix among bivalves and Cerethideopsilla cingulata, Bullia vittata, Nassarious stolatus, Indothias lacera, Natica tigrina, Turritella attenuata were from the gastropods. Occurrence of a large number of marine taxa is most probably associated with the opening of new lagoon during 1st August 2008.     

Comprehensive analysis of fungal diversity and enzyme activity in <Emphasis Type="Italic">nuruk</Emphasis>, a Korean fermenting starter,for acquiring useful fungi

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3–1.8 × 10⁸ CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.