Identification of Chitin in Pliocene Fungi Using Py-GC × GC-TOFMS: Potential Implications for the Study of the Evolution of the Fungal Clade in Deep Time.

Molecular dating estimates the origin of the fungal clade to the Pre-Cambrian. Yet, the oldest unambiguous fungal fossils date to the Ordovician and show remarkable diversity and organizational development. Recent studies have suggested that the dates for the emergence of fungi in the fossil record may be pushed back to the Proterozoic. However, the nonspecificity of the methods used in those studies necessitates the employment of a wider variety of analytical techniques that can independently verify the presence of chitin, a crucial prerequisite in the assignment of fungal affinity, particularly of putative fossils from the Pre-Cambrian. In this paper, we propose Py-GC × GC-TOFMS as an example of one such technique. We analyze fungal fossils from the Pliocene. We find that a suite of N-bearing compounds are present in the pyrolysis products of these fossils, from which we suggest that 3-acetamidopyrones and their methylated homologues can serve as specific pyrolytic markers for chitin. We discuss both how this technique can potentially be used to differentiate between biopolymers, including those similar to chitin such as peptidoglycan, and the potential implications of identifying such markers in fossils from deep time. We conclude that Py-GC × GC-TOFMS is a promising technique that can potentially be used alongside, or independent of, staining methods to detect the presence of chitin in fossils.

Evidence of the oldest extant vascular plant (horsetails) from the Indian Cenozoic.

Equisetum (Equisetaceae) has long been a focus of attention for botanists and palaeontologists because, given its extensive and well-documented fossil record, it is considered the oldest extant vascular plant and a key element in understanding vascular plant evolution. However, to date, no authentic fossil evidence of Equisetum has been found from the Indian Cenozoic. Here, we describe a new fossil species, namely, E. siwalikum sp. nov., recovered from the middle Siwalik (Late Miocene) sediments of Himachal Pradesh, western Himalaya. We identified fossil specimens based on morphological and epidermal characters. In addition, X-Ray diffraction (XRD) analysis was used to determine the mineral composition of compressed stems of Equisetum. The close affinity of our recovered Siwalik fossils to Equisetum is supported by the presence of both macromorphological and epidermal characters. Because Equisetum generally grows in wet conditions around water reservoirs, our findings indicate that the fossil locality was humid and surrounded by swamp and lowland regions during deposition. Ample fossil evidence indicates that this sphenopsid once existed in the western Himalaya during the Siwalik period. However, at present Equisetum is confined to a particular area of our fossil locality, probably a consequence of severe environmental changes coupled with competition from opportunistic angiosperms. Our discovery of Equisetum fossils in appreciable numbers from the Siwalik sediments of the Himachal Himalayas is unique and constitutes the first reliable recognition of Equisetum from the Indian Cenozoic.

Marginal leaf galls on Pliocene leaves from India indicate mutualistic behavior between Ipomoea plants and Eriophyidae mites.

We report a new type of fossil margin galls arranged in a linear series on dicot leaf impressions from the latest Neogene (Pliocene) sediments of the Chotanagpur Plateau, Jharkhand, eastern India. We collected ca. 1500 impression and compression leaf fossils, of which 1080 samples bear arthropod damage referable to 37 different damage types (DT) in the 'Guide to Insect (and Other) Damage Types in Compressed Plant Fossils'. A few leaf samples identified as Ipomoea L. (Convolvulaceae) have specific margin galls that do not match any galling DT previously described. This type of galling is characterized by small, linearly arranged, irregular, sessile, sub-globose, solitary, indehiscent, solid pouch-galls with irregular ostioles. The probable damage inducers of the present galling of the foliar margin might be members of Eriophyidae (Acari). The new type of gall suggests that marginal gall-inducing mites on leaves of Ipomoea did not change their host preference at the genus level since the Pliocene. The development of marginal leaf galling in Ipomoea is linked to extrafloral nectaries that do not offer protection against arthropod galling but indirectly protect the plant against herbivory from large mammals.

Coryphoid palms from the K-Pg boundary of central India and their biogeographical implications: Evidence from megafossil remains.

Ten palm leaf impressions are documented from the latest Maastrichtian (late Cretaceous) to early Danian (earliest Paleocene) sediments (K-Pg, c. 66-64 Ma) of the Mandla Lobe of the Deccan Inter-trappean Beds, Madhya Pradesh, central India. The palmate leaf shape along with a definite well-preserved costa support their placement in the subfamily Coryphoideae of the family Arecaceae. We place all recovered palm leaf specimens in the fossil genus Sabalites, report seven species of coryphoid palms and describe two new species namely, Sabalities umariaensis sp. nov. and Sabalites ghughuaensis sp. nov. The fossils indicate that coryphoid palms were highly diverse in central India by the latest Cretaceous. These and earlier reported coryphoid palm fossils from the same locality indicate that they experienced a warm and humid tropical environment during the time of deposition. These discoveries confirm the presence of a diversity of Coryphoideae in Gondwana prior to the India-Eurasia collision and provide information about coryphoid biogeographical history over geological time. Based on megafossil remains, we trace coryphoid palm migration pathways from India to mainland Southeast (SE) Asia and other parts of Asia after the docking of the Indian subcontinent with Eurasia early in the Paleogene.

Siwalik plant megafossil diversity in the Eastern Himalayas: A review.

The Eastern Himalayas are renowned for their high plant diversity. To understand how this modern botanical richness formed, it is critical to investigate past plant biodiversity preserved as fossils throughout the eastern Himalayan Siwalik succession (middle Miocene-early Pleistocene). Here, we present a summary of plant diversity records that document Neogene floristic and climate changes. We do this by compiling published records of megafossil plant remains, because these offer better spatial and temporal resolution than do palynological records. Analyses of the Siwalik floral assemblages based on the distribution of the nearest living relative taxa suggest that a tropical wet evergreen forest was growing in a warm humid monsoonal climate at the deposition time. This qualitative interpretation is also corroborated by published CLAMP (Climate Leaf Analysis Multivariate Program) analyses. Here, we also reconstruct the climate by applying a new common proxy WorldClim2 calibration. This allows the detection of subtle climate differences between floral assemblages free of artefacts introduced by using different methodologies and climate calibrations. An analysis of the Siwalik floras indicates that there was a gradual change in floral composition. The lower Siwalik assemblages provide evidence of a predominance of evergreen elements. An increase in deciduous elements in the floral composition is noticed towards the close of the middle Siwalik and the beginning of the upper Siwalik formation. This change reflects a climatic difference between Miocene and Plio-Pleistocene times. This review helps us to understand under what paleoenvironmental conditions plant diversity occurred and evolved in the eastern Himalayas throughout the Cenozoic.

The diagenetic fate of collagen as revealed by analytical pyrolysis of fossil fish scales from deep time.

The mechanism of protein degradation has remained a topic of debate (specifically concerning their preservation in deep time), which has recently been invigorated due to multiple published reports of preservation ranging from Miocene to the Triassic that potentially challenge the convention that protein preservation beyond the Cenozoic is extremely uncommon or is expected to be absent altogether, and thus have attracted skepticism. In this paper, we analyze fossil fish scales from the Cretaceous, Jurassic, and Triassic using comprehensive pyrolysis gas chromatography coupled with time-of-flight mass spectrometry and compare the pyrolytic products so obtained with a well-preserved fish scale from Late Pliocene, in an attempt to better understand the effects of diagenesis on protein degradation at the molecular level through deep time. We find that the Pliocene fish scale displays a large number of N-bearing pyrolytic products, including abundant substituted cyclic 2,5-diketopiperazines (2,5-DKPs) which are diagnostic products of peptide and amino acid pyrolysis. We identify N-bearing compounds in the Mesozoic fish scales-however, among the 2,5-DKPs that were identified in the Pliocene scale, only diketodipyrrole (or cyclo (Pyr-Pyr)) is present in the Mesozoic scales. We discuss the implications of N-bearing pyrolytic products with emphasis on 2,5-DKPs in geological samples and conclude that the discrepancy in abundance and variety of N-bearing products between Pliocene and Mesozoic scales indicates that the protein component in the latter has been extensively diagenetically altered, while a suite of DKPs such as in the former would imply stronger evidence to indicate preservation of protein. We conclude that analytical pyrolysis is an effective tool for detecting preservation of intact proteins, as well as for providing insights into their degradation mechanisms, and can potentially be utilized to assign proteinaceous origin to a fossil sample of unknown affinity.

A novel fossil-species of Meliolinites Selkirk (fossil Meliolaceae) and its life cycle stages associated with an angiosperm fossil leaf from the Siwalik (Mio-Pliocene) of Bhutan sub-Himalaya.

Here, we report the in-situ occurrence of a new fossil-species of Meliolinites (fossil Meliolaceae), Meliolinites bhutanensis sp. nov. on the cuticle fragments of a compressed angiosperm dicot leaf recovered from the middle Siwalik (Formation II: latest Miocene to Pliocene) of Bhutan, eastern Himalaya. This unique foliicolous new fossil fungal species features well-preserved mycelia consisting of superficial, brown to dark brown, septate, thick-walled, branching hyphae with bi-cellular appressoria, unicellular phialides, and a characteristic long, slightly curved hyphal seta. The web-like, brown to dark brown fungal colonies also include globose to sub-globose, dark brown ascomata, and oblong to broadly cylindrical, 5-celled, 4-septate, brown to dark brown, mature ascospores. As almost all features of different stages in the life cycle (ascospores, mature germinating ascospores, superficial lateral hyphae, hyphal seta, hyphopodia, mycelial colony, and ascomata) of this new fossil-species are found, we have proposed the first time a possible life cycle of fossil-species of Meliolaceae. The in-situ evidence of M. bhutanensis on the host leaf cuticle indicates the possible existence of a host-ectoparasite relationship in Bhutan sub-Himalaya's ancient warm and humid tropical evergreen forest during the deposition. So, M. bhutanensis might have thrived generally under warm and humid climate conditions for its growth and development in the Mio-Pliocene time, which is in conformity with our recently published quantitative climatic data by CLAMP (Climate Leaf Analysis Multivariate Program) analysis.

A comprehensive calibrated phytolith based climatic index from the Himalaya and its application in palaeotemperature reconstruction.

Past climate reconstructions from palaeoecological records require an understanding of relationships between modern vegetation and climate. Phytoliths are being used widely to reconstruct variations in C3/C4 grasses in the past vegetation and corresponding climate. However, little understanding is available on their relationships with the climate driver(s). Even though, the driver(s) regulating C3/C4 grass distributions vary regionally, while reconstructing the past distributions, a grass phytolith-based climatic index (Ic) has often been found to be used globally without assessing its regional consistency. In the Himalaya, the working potential of Ic has proven to be unsatisfactory when compared to other regions of the globe. To improve the efficacy of Ic, we have identified the redundant grass phytolith morphs and revised it by including four exclusive C3-grass indicator morphotypes (bilobate trapezoidal, bilobate scooped, saddle tall and saddle plateaued) to the existing Ic calculation. Thus, a new climatic index, revised Ic (rIc) is proposed in this article. We have compared the rIc with modern climate variables and a relationship with mean annual temperature (MAT) is established with statistical validation. To assess the working potential of the proposed calibration function in the past temperature reconstructions, we have estimated the late Holocene MAT variations in the Himalaya using rIc. We infer that in the mountainous regions like the Himalaya, even with irregular precipitation distribution, variability in C3/C4 grass distributions and their phytolith spectra seem to be a primary function of temperature. Further, we recommend that rIc can be satisfactorily used to reconstruct past temperature variations in the Himalaya and similar mountainous regions where soil water availability is not a limiting factor.

Evidence of fungal decay in petrified legume wood from the Neogene of the Bengal Basin, India.

Silicified fossil legume woods of Cynometroxylon Chowdhury & Ghosh collected from the Neogene (late Miocene) sediments of the Bengal Basin, eastern India, exhibit fungal decay seldom found in the fossil record. The wood possesses numerous perforate areas on the surface that seem to be the result of extensive fungal activity. In transverse section, the decayed areas (pockets) appear irregular to ellipsoidal in outline; in longitudinal section these areas of disrupted tissue are somewhat spindle-shaped. Individual pockets are randomly scattered throughout the secondary xylem or are restricted to a narrow zone. The aforesaid patterns of decay in fossil wood show similarities with that of white rot decay commonly produced by higher fungi, specifically basidiomycetes and ascomycetes. The host fossil wood harbors abundant ramifying and septate fungal hyphae with knob like swellings similar to pseudoclamps in basidiomycetes, and three-celled conidia-like reproductive structures. This record expands our current knowledge of wood decaying fungi-host plant interaction in the Neogene tropical forests of Peninsular India.

Occurrence of Phoma Sacc. in the phyllosphere of Neogene Siwalik forest of Arunachal sub-Himalaya and its palaeoecological implications.

The present study reports in situ occurrence of two new epiphyllous fungal species of Phomites (comparable to modern genus Phoma Sacc.) on angiospermic leaf remains recovered from the Siwalik sediments (middle Miocene to early Pleistocene) of Arunachal Pradesh, eastern Himalaya. We describe two new species i.e. Phomites siwalicus Vishnu, Khan et Bera S, sp. nov. and Phomites neogenicus Vishnu, Khan et Bera S, sp. nov. on the basis of structural details of pycnidia. The pycnidium is a globose or slightly lens-shaped, ostiolate with a collar layer consisting of thick walled cells, sunken in leaf cuticle, with one-celled conidiospores and short-ampulliform conidiogenous cells. Host leaves resemble to those of extant Dipterocarpus C. F. Gaertn., Shorea Roxb. ex C. F. Gaertn. (Dipterocarpaceae), Dysoxylum Blume (Meliaceae), and Poaceae Barnhart. In situ occurrence of two Phomites morphotypes on the said leaf remains suggests a possible host-parasite interaction in the moist evergreen forest of Arunachal sub-Himalaya during Mio-Pleistocene period. The occurrence of Phomites in appreciable numbers indicates a humid climate favored by high rate of precipitation during Siwalik sedimentation, which is also consistent with our previously published climatic data obtained from the study of the macroscopic plant remains.

Phytochemical and genetic analyses of ancient cannabis from Central Asia.

The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.

Soral crypsis: protective mimicry of a coccid on an Indian fern.

Herbivory with crypsis is not well documented in ferns. The present record of cryptic coloration of coccid Saissetia filicum Boisduval (Homoptera: Coccidae) to the sori of a fern species Asplenium nidus L. (Aspleniaceae) is unique. Predatory beetles (Jauravia sp., Coleoptera: Coccinellidae) that feed on the coccids, are suggested to be selective pressure for the development of the present homopteran soral crypsis. A higher rate of effective predation is noticed in the vegetative leaves than the fertile leaves. Aggressive ants were found harvesting honeydew secretions from the coccids and defending the trophobionts as well as the host fern from their natural enemies. In addition, a possible three-way mutualistic relationship among the coccids, its host fern and the tending ant is suggested. Differential numbers of coccids on vegetative and fertile leaves is correlated with their phenol content and degree of predation by beetles. Such coloration mimicry by the coccids may enable them to obtain the necessary blend of sorus of the host fern needed to evade beetle detection and attack.

A new insight into Cannabis sativa (Cannabaceae) utilization from 2500-year-old Yanghai Tombs, Xinjiang, China.

A cache of shoots, leaves and fruits dated by (14)C at 2500 years B.P. were unearthed in the Yanghai Tombs, Turpan District in Xinjiang, China. By comparing the morphological and anatomical characteristics of the plant remains found in the tomb and specimens of modern plants, it is shown that the remains belong to Cannabis. Based on the shamanistic background of the deceased man and ancient customs, it is assumed that the Cannabis was utilized for ritual/medicinal purposes.

Depositional Environment of Mio-Pliocene Siwalik Sedimentary Strata from the Darjeeling Himalayan Foothills, India: A Palynological Approach.

A rich and diverse palynoassemblage recovered from the Churanthi River section (26°53' 59.3" N, 88°34' 17.2" E), Darjeeling foothills Eastern Himalaya, has yielded 87 species assigned to 69 genera. The palynoassemblage is rich in angiosperm taxa (45.63%) followed by gymnosperms (0.45%), pteridophytes (18.49%) and fungal remains (23.88%). Based on their nearest living relatives, a wet evergreen to semi-evergreen forest under a humid tropical to sub-tropical environment during the Mio-Pliocene age has been suggested. A lot of angiosperms such as Palaeosantalaceaepites, Araliaceoipollenites, Malvacearampollis, Zonocostites, Neocouperipollis, Dicolpopollis, Palmidites, Palmaepollenites, isolated salt glands of mangrove plant leaves (Heliospermopsis) and Mediaverrunites type of fungal spores, along with ichnofossils like Planolites, Palaeophycus, Skolithos, Rosselia, Ophiomorpha and Teichichnus associated with rippled mudstone-siltstone suggest an environment strongly influenced by brackish water. Primary sedimentary structures in the associated strata indicate strong wave agitation common in shallow marine setting. Some high elevation components (5.14%) such as Alnipollenites, cf. Corylus (Betulaceae), Juglanspollenites, Engelhardtioipollenites (Juglandaceae), Quercoides, Cupuliferoidaepollenites, Lithocarpus, Castanopsis (Fagaceae), Abietineaepollenites (Pinaceae) represent hinterland vegetation possibly transported to the prograding deltaic coastline by the rivers. Reworked palynotaxa (Striatopodocarpites sp., Striatites sp., Faunipollenites sp., Circumstriatites sp., Crescentipollenites sp., Cuneatisporites sp., Parasaccites sp., Scheuringipollenites sp., Rhizomaspora sp., Marsupipollenites sp., Lophotriletes sp.) of Permian age have also been recorded in the palynoassemblage (11.55%) indicating the abundance of Permian Gondwana strata in the source area.

Prospects of apicultural entrepreneurship in coastal districts of eastern India: a melissopalynological evaluation.

A melissopalynological analysis of fifty-one natural honey samples (twenty four spring, fifteen summer and twelve winter) collected during 2010-2011 from two east-coastal districts (20(0)20/ to 22(0)11/ N, 82(0)39/ to 87(0)01/ E) of Orissa, India was performed. Out of 37 unifloral samples found 25 were contributed by Apis cerana indica, seven by A. dorsata and the remaining five by A. florea. Out of 14 multifloral samples five were contributed by A. cerana indica, five by A. dorsata and the remaining four by A. florea. Principal component analysis confirmed the palynological classification of the unifloral honey samples. Eighty-two bee-plant taxa belonging to forty four families were recovered. The predominant nectariferous taxa of the spring season were Acanthus ilicifolius, Avicennia marina, Bruguiera gymnorrhiza, Cocos nucifera, Eucalyptus globulus, Phoenix paludosa, Pongamia pinnata, Prosopis juliflora, Sonneratia apetala and Syzygium cumini. In the summer the predominant nectariferous taxa were Borassus flabellifer, C. nucifera, E. globulus, Syzygium cumini, Terminalia arjuna, Aegiceras corniculatum, P. paludosa and Sonneratia apetala while those of the winter were Brassica nigra, Coriandrum sativum, Zizyphus jujuba, Alstonia scholaris, E. globulus and Bruguiera gymnorrhiza. Very low (<0.09) HDE/P for 98% of the samples and absence of toxic palynotaxa assure that these honeys are suitable for human consumption. Quite extended honey flow period with spring and summer as best forage seasons for the honeybees and occurrence of 82% of these honeys with APC Group II, III and IV justify the sustainability of the present study area for establishing moderate to large-scale apicultural entrepreneurship. This should improve the socio-economic status of the people of this region.

Pollen and phytoliths from fired ancient potsherds as potential indicators for deciphering past vegetation and climate in Turpan, Xinjiang, NW China.

It is demonstrated that palynomorphs can occur in fired ancient potsherds when the firing temperature was under 350°C. Pollen and phytoliths recovered from incompletely fired and fully fired potsherds (ca. 2700 yrs BP) from the Yanghai Tombs, Turpan, Xinjiang, NW China can be used as potential indicators for reconstructing past vegetation and corresponding climate in the area. The results show a higher rate of recovery of pollen and phytoliths from incompletely fired potsherds than from fully fired ones. Charred phytoliths recovered from both fully fired and incompletely fired potsherds prove that degree and condition of firing result in a permanent change in phytolith color. The palynological data, together with previous data of macrobotanical remains from the Yanghai Tombs, suggest that temperate vegetation and arid climatic conditions dominated in the area ca. 2700 yrs BP.