Morphological and Ultrastructural Features of Selected Epidendroideae Pollen Dispersal Units and New Insights into Their Chemical Nature.

Orchidaceae display enormous diversity in their flower morphology, which is particularly evident in their pollen dispersal units (pollinia, pollinaria). The packaging of pollen by elastoviscin leads to a great diversity of these morphologically and structurally complex pollen units. Despite being one of the most diverse angiosperm families, the available palynological data on orchids remain limited and sometimes contradicting. This study provides new insights into the pollen morphology and ultrastructure of five orchid species from the subfamily Epidendroideae, using combined light, scanning electron, and transmission electron microscopy. The aim was to compare the morphology and ultrastructure of pollen dispersal units and to elucidate the chemical nature of the pollen wall layers and of elastoviscin. Our combined light and electron microscopy investigation demonstrated the presence of six tetrad types even within a single pollinium, which is unique for orchids. The application of different staining methods confirmed the assumed lipidic nature of elastoviscin and the differences in its contrast and ultrastructure suggest a mixture of sticky materials with dissimilar chemical compositions. This study affirmed that sporopollenin is mostly restricted to the outer pollen grains of peripheral tetrads in compact and sectile pollinia, while inner tetrads exhibit highly reduced non-sporopollenin pollen walls.

Origin and Early Evolution of Hydrocharitaceae and the Ancestral Role of Stratiotes.

The combined morphological features of Stratiotes (Hydrocharitaceae) pollen, observed with light and electron microscopy, make it unique among all angiosperm pollen types and easy to identify. Unfortunately, the plant is (and most likely was) insect-pollinated and produces relatively few pollen grains per flower, contributing to its apparent absence in the paleopalynological record. Here, we present fossil Stratiotes pollen from the Eocene of Germany (Europe) and Kenya (Africa), representing the first reliable pre-Pleistocene pollen records of this genus worldwide and the only fossils of this family discovered so far in Africa. The fossil Stratiotes pollen grains are described and compared to pollen from a single modern species, Stratiotes aloides L. The paleophytogeographic significance and paleoecological aspects of these findings are discussed in relation to the Hydrocharitaceae fossil records and molecular phylogeny, as well as the present-day distribution patterns of its modern genera.

First evidence of a monodominant (Englerodendron, Amherstieae, Detarioideae, Leguminosae) tropical moist forest from the early Miocene (21.73 Ma) of Ethiopia.

Many tropical wet forests are species-rich and have relatively even species frequency distributions. But, dominance by a single canopy species can also occur in tropical wet climates and can remain stable for centuries. These are uncommon globally, with the African wet tropics supporting more such communities than the Neotropics or Southeast Asia. Differences in regional evolutionary histories are implied by biogeography: most of Africa's monodominance-forming species are Amherstieae-tribe legumes; monodominance in Neotropical forests occur among diverse taxonomic groups, often legumes, but rarely Amherstieae, and monodominance in Southeast Asian forests occurs mostly among Dipterocarpaceae species. African monodominant forests have been characterized ecologically and taxonomically, but their deep-time history is unknown despite their significant presence and bottom-up ecological influence on diversity. Herein we describe fossil leaflets of Englerodendron mulugetanum sp. nov., an extinct species of the extant genus Englerodendron (Berlinia Clade, Amherstieae, Detarioideae) from the 21.73 Ma Mush Valley site in Ethiopia. We also document a detailed study of associated legume pollen, which originate from a single taxon sharing characters with more than one extant descendant. Taxonomically, the pollen is most comparable to that from some extant Englerodendron species and supports a likely affiliation with the Englerodendron macrofossils. The Mush Valley site provides the first fossil evidence of a monodominant tropical forest in Africa as represented by leaflets and pollen. Previous studies documented >2400 leaves and leaflets from localities at six stratigraphic levels spanning 50,000-60,000 years of nearly continuous deposition within seven meters of section; all but the basal level contain ≥ 50% E. mulugetanum leaflets. Modern leaf litter studies in African mixed vs. monodominant forests indicates the likelihood of monodominance in the forests that surrounded the Mush paleolake, particularly after the basal level. Thus, we provide an early case for monodominance within the Amherstieae legumes in Africa.

Large giraffids (Mammalia, Ruminantia) from the new late Miocene fossiliferous locality of Kemiklitepe-E (Western Anatolia, Turkey).

Kemiklitepe is a well-known locality with four recognised fossiliferous horizons, KTA to KTD, which have yielded a plethora of mammalian remains. Previous taxonomic studies indicate the presence of three giraffid taxa: Samotherium major and Palaeotragus rouenii from the uppermost three horizons, KTA, KTB and KTC, as well as Palaeotragus rouenii and Samotherium? sp. from the lowermost KTD horizon. In this study a new locality, Kemiklitepe-E, is presented for the first time. Kemiklitepe-E is located approximately 350 m NW of the classic Kemiklitepe locality. The fossiliferous sedimentary rocks at Kemiklitepe-E occur at the same stratigraphic level as localities KTA, KTB and KTC. The preliminary faunal list includes representatives of Proboscidea, Chalicotheriidae, Equidae, Bovidae and Giraffidae. Comprehensive descriptions and comparisons of the Kemiklitepe-E Giraffidae specimens suggest the co-occurrence of two large giraffids: Samotherium major and Helladotherium duvernoyi. Samotherium major, previously documented from this region, is the most common taxon at Kemiklitepe. Helladotherium duvernoyi is rare at Kemiklitepe and here reported for the first time. The two taxa coexisted during the middle Turolian in Greece and Western Anatolia. In addition, it is suggested that specimens of Samotherium? sp described from KTD possibly belong to Samotherium neumayri. Based on the stratigraphic position of fossiliferous rocks, as well as the faunal data presented herein, the newly discovered locality is considered to be of middle Turolian (MN12) age.

How to extract and analyze pollen from internal organs and exoskeletons of fossil insects?

This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison. For complete details on the use and execution of this protocol, please refer to Wappler et al. (2015), Ulrich and Grímsson (2020), and Wedmann et al. (2021).

Hagenia from the early Miocene of Ethiopia: Evidence for possible niche evolution?

Fossil pollen believed to be related to extant Hagenia abyssinica were discovered in the early Miocene (21.73 Ma) Mush Valley paleoflora, Ethiopia, Africa. Both the fossil and extant pollen grains of H. abyssinica were examined with combined light microscopy, scanning electron microscopy, and transmission electron microscopy to compare the pollen and establish their relationships. Based on this, the fossil pollen grains were attributed to Hagenia. The presence of Hagenia in the fossil assemblage raises the questions if its habitat has changed over time, and if the plants are/were wind pollinated. To shed light on these questions, the morphology of extant anthers was also studied, revealing specialized hairs inside the anthers, believed to aid in insect pollination. Pollen and anther morphology are discussed in relation to the age and origin of the genus within a molecular dated phylogenetic framework, the establishment of complex topography in East Africa, other evidence regarding pollination modes, and the palynological record. The evidence presented herein, and compiled from the literature, suggests that Hagenia was an insect-pollinated lowland rainforest element during the early Miocene of the Mush Valley. The current Afromontane habitat and ambophilous (insect and wind) pollination must have evolved in post-mid-Miocene times.

The last meal of an Eocene pollen-feeding fly.

One of the most important trophic interactions today is that between insects and their floral hosts. This biotic association is believed to have been critical to the radiation of flowering plants and many pollinating insect lineages over the last 120 million years (Ma). Trophic interactions among fossil organisms are challenging to study, and most inferences are based on indirect evidence. Fossil records providing direct evidence for pollen feeding, i.e., fossil stomach and gut contents, are exceptionally rare.1,2 Such records have the potential to provide information on aspects of animal behavior and ecology as well as plant-animal interactions that are sometimes not yet recognized for their extant relatives. The dietary preferences of short-proboscid nemestrinids are unknown, and pollinivory has not been recorded for extant Nemestrinidae.3 We analyzed the contents of the conspicuously swollen abdomen of an ca. 47.5 Ma old nemestrinid fly of the genus Hirmoneura from Messel, Germany, with photogrammetry and state-of-the-art palynological methods. The fly fed on pollen from at least four plant families-Lythraceae, Vitaceae, Sapotaceae, and Oleaceae-and presumably pollinated flowers of two extant genera, Decodon and Parthenocissus. We interpret the feeding and foraging behavior of the fly, reconstruct its preferred habitat, and conclude about its pollination role and importance in paratropical environments. This represents the first evidence that short-proboscid nemestrinid flies fed, and possibly feed to this day, on pollen, demonstrating how fossils can provide vital information on the behavior of insects and their ecological relationships with plants.

Palaeodietary traits of large mammals from the middle Miocene of Gracanica (Bugojno Basin, Bosnia-Herzegovina).

Recent excavations at the Gracanica coal mine (Bugojno Basin, Bosnia-Herzegovina) have unearthed numerous skeletal parts of fossil vertebrates, including a noteworthy collection of mammalian remains. Previous palaeoecological investigations of the Dinarides Lake System were established using stratigraphical, palaeofloral, and malacological data. However, large mammal remains have so far not been used to reconstruct the terrestrial palaeoenvironment of this important fossil ecosystem. Here, the palaeodietary preferences of large mammals were investigated, using a multiproxy approach by employing dental microwear and dental mesowear analysis, in order to provide new perspectives on the terrestrial palaeoecology of the Dinarides Lake System. The dental microwear of all available adult mammalian teeth was analysed. Dental mesowear analysis was employed for ungulate and proboscidean taxa, using mesowear scores and mesowear angles, respectively. The analysis reveals the presence of browsing, "dirty browsing", and mixed-feeding herbivorous taxa, with seasonal fruit, or even grass intake. Additionally, the analysis of the carnivores suggests the presence of hyaena- and cheetah-like hypercarnivores, as well as generalists. The palaeodietary traits of the fossil mammals suggest a closed canopy-like environment, which is supported by the fossil plant assemblage. Palaeopalynological data confirm the omnipresence of fleshy fruit-bearing plants, herbaceous taxa, as well as grasses, which justifies the seasonal fruit browsing, the common "dirty browsing", and the occasional grazing behaviour visualized for some of the fossil mammals from Gracanica.

Combined LM and SEM study of the middle Miocene (Sarmatian) palynoflora from the Lavanttal Basin, Austria: Part V. Magnoliophyta 3 - Myrtales to Ericales.

The continued investigation of the middle Miocene palynoflora from the Lavanttal Basin reveals numerous additional angiosperm taxa. The Myrtales to Ericales pollen record documented here comprises 46 different taxa belonging to Onagraceae (Ludwigia), Ericaceae (Craigia, Reevesia, Tilia), Anacardiaceae (Pistacia), Rutaceae (Zanthoxylum), Sapindaceae (Acer), Santalaceae (Arceuthobium), Amaranthaceae, Caryophyllaceae, Polygonaceae (Persicaria, Rumex), Cornaceae (Alangium, Cornus, Nyssa), Ebenaceae (Diospyros), Ericaceae (Andromeda, Arbutus, Empetrum, Erica), Sapotaceae (Pouteria, Sideroxylon), Styracaceae (Rehderodendron) and Symplocaceae (Symplocos). Köppen signatures of potential modern analogues of the additional fossil woody elements confirm the hypothesis of a subtropical (Cfa, Cwa) climate at lower elevations and subsequent transition into a temperate climate with altitudinal succession (Cfa → Cfb/Dfa → Dfb; Cwa → Cwb → Dwb-climate). The fossil plants represent different vegetation units, from wetland lowlands to well-drained montane forests. Many of the fossil taxa have potential modern analogues that can be classified as nemoral and/or meridio-nemoral and/or semihumid-meridional vegetation elements. New is the recognition of oreotropical elements, which are direct indicators for a substantial altitudinal gradient.

The single-grain method: adding TEM to the equation.

An advanced protocol to prepare single extant and fossil pollen grains for transmission electron microscopy (TEM) analysis allows for the fast recovery of data on the ultrastructure of pollen/spores. The protocol is easy to apply and less time consuming than previous methods. The 'loss' of pollen grains and pollen that is 'difficult to locate' within the embedding material is avoided, and each single pollen grain can be prepared successfully for TEM analysis. This preparation method is meant as an addition to the single-grain method using combined light and scanning electron microscopy to investigate dispersed fossil pollen grains developed by Dr Reinhard Zetter in the late 1980s.

Origin and divergence of Afro-Indian Picrodendraceae: linking pollen morphology, dispersal modes, fossil records, molecular dating and paleogeography.

The pantropical Picrodendraceae produce mostly spheroidal to slightly oblate, echinate pollen grains equipped with narrow circular to elliptic pori that can be hard to identify to family level in both extant and fossil material using light microscopy only. Fossil pollen of the family have been described from the Paleogene of America, Antarctica, Australia, New Zealand, and Europe, but until now none have been reported from Afro-India. Extant pollen described here include representatives from all recent Picrodendraceae genera naturally occurring in Africa and/or Madagascar and south India and selected closely related tropical American taxa. Our analyses, using combined light microscopy and scanning electron microscopy, show that pollen of the Afro-Indian genera encompass three morphological types: Type 1, comprising only Hyaenanche; Type 2, including Aristogeitonia, Mischodon, Oldfieldia and Voatamalo; Type 3, comprising the remaining two genera, Androstachys and Stachyandra. Based on the pollen morphology presented here it is evident that some previous light microscopic accounts of spherical and echinate fossil pollen affiliated with Arecaceae, Asteraceae, Malvaceae, and Myristicaceae from the African continent could belong to Picrodendraceae. The pollen morphology of Picrodendraceae, fossil pollen records, a dated intra-familial phylogeny, seed dispersal modes, and the regional Late Cretaceous to early Cenozoic paleogeography, together suggest the family originated in the Americas and dispersed from southern America across Antarctica and into Australasia. A second dispersal route is believed to have occurred from the Americas into continental Africa via the North Atlantic Land Bridge and Europe.

Sclerosperma fossils from the late Oligocene of Chilga, north-western Ethiopia.

The palm family, Arecaceae, is notoriously depauperate in Africa today, and its evolutionary, paleobiogeographic, and extinction history there are not well documented by fossils. In this article we report the pollen of two new extinct species of the small genus, Sclerosperma (Arecoideae), from a late Oligocene (27-28 Ma) stratum exposed along the Guang River in Chilga Wereda of north-western Ethiopia. The pollen are triporate, and the two taxa can be distinguished from each other and from modern species using a combination of light and scanning electron microscopy, which reveals variations in the finer details of their reticulate to perforate exine sculpture. We also report a palm leaf fragment from a stratum higher in the same section that is in the Arecoideae subfamily, and most likely belongs to Sclerosperma. The implications of these discoveries for the evolutionary history of this clade of African arecoid palms is that their diversification was well underway by the middle to late Oligocene, and they were much more widespread in Africa at that time than they are now, limited to West and Central Africa. Sclerosperma exhibits ecological conservatism, as today it occurs primarily in swamps and flooded forests, and the sedimentology of the Guang River deposits at Chilga indicate a heterogeneous landscape with a high water table. The matrix containing the fossil pollen is lignite, which itself indicates standing water, and a variety of plant macrofossils from higher in the section have been interpreted as representing moist tropical forest or seasonally inundated forest communities.

Pollen morphology of the African Sclerosperma (Arecaceae).

Three currently accepted Sclerosperma species appear to produce four different pollen morphologies. Sclerosperma mannii and S. walkeri pollen share the same distinct reticulate sculpture, but S. profizianum produces three different pollen types (microreticulate, fossulate, and perforate). The pollen morphology suggests that S. mannii and S. walkeri are sister taxa of the same intrageneric lineage. The pollen diversity observed in S. profizianum suggests (a) this taxon is unique regarding its pollen diversity despite being a non-heterostylous plant or (b) that circumscription of S. profizianum as a species may well be in the need of redefinition.

The first Loranthaceae fossils from Africa.

An ongoing re-investigation of the early Miocene Saldanha Bay (South Africa) palynoflora, using combined light and scanning electron microscopy (single grain method), is revealing several pollen types new to the African fossil record. One of the elements identified is Loranthaceae pollen. These grains represent the first and only fossil record of Loranthaceae in Africa. The fossil pollen grains resemble those produced by the core Lorantheae and are comparable to recent Asian as well as some African taxa/lineages. Molecular and fossil signals indicate that Loranthaceae dispersed into Africa via Asia sometime during the Eocene. The present host range of African Loranthaceae and the composition of the palynoflora suggest that the fossil had a range of potential host taxa to parasitise during the early Miocene in the Saldanha Bay region.

Evolution of pollen morphology in Loranthaceae.

Earlier studies indicate a strong correlation of pollen morphology and ultrastructure with taxonomy in Loranthaceae. Using high-resolution light microscopy and scanning electron microscopy imaging of the same pollen grains, we document pollen types of 35 genera including 15 studied for the first time. Using a molecular phylogenetic framework based on currently available sequence data with good genus-coverage, we reconstruct trends in the evolution of Loranthaceae pollen and pinpoint traits of high diagnostic value, partly confirming earlier intuitive hypotheses based on morphological observations. We find that pollen morphology in Loranthaceae is strongly linked to phylogenetic relationships. Some pollen types are diagnostic for discrete genera or evolutionary lineages, opening the avenue to recruit dispersed fossil pollen as age constraints for dated phylogenies and as independent data for testing biogeographic scenarios; so far based exclusively on modern-day data. Correspondences and discrepancies between palynological and molecular data and current taxonomic/systematic concepts are identified and suggestions made for future palynological and molecular investigations of Loranthaceae.

Eocene Loranthaceae pollen pushes back divergence ages for major splits in the family.

BACKGROUND: We revisit the palaeopalynological record of Loranthaceae, using pollen ornamentation to discriminate lineages and to test molecular dating estimates for the diversification of major lineages. METHODS: Fossil Loranthaceae pollen from the Eocene and Oligocene are analysed and documented using scanning-electron microscopy. These fossils were associated with molecular-defined clades and used as minimum age constraints for Bayesian node dating using different topological scenarios. RESULTS: The fossil Loranthaceae pollen document the presence of at least one extant root-parasitic lineage (Nuytsieae) and two currently aerial parasitic lineages (Psittacanthinae and Loranthinae) by the end of the Eocene in the Northern Hemisphere. Phases of increased lineage diversification (late Eocene, middle Miocene) coincide with global warm phases. DISCUSSION: With the generation of molecular data becoming easier and less expensive every day, neontological research should re-focus on conserved morphologies that can be traced through the fossil record. The pollen, representing the male gametophytic generation of plants and often a taxonomic indicator, can be such a tracer. Analogously, palaeontological research should put more effort into diagnosing Cenozoic fossils with the aim of including them into modern systematic frameworks.

Tiny pollen grains: first evidence of Saururaceae from the Late Cretaceous of western North America.

BACKGROUND: The Saururaceae, a very small family of Piperales comprising only six species in four genera, have a relatively scanty fossil record outside of Europe. The phylogenetic relationships of the four genera to each other are resolved, with the type genus Saururus occurring in both eastern North America and East Asia. No extant species occurs in western Eurasia. The most exceptional find so far has been an inflorescence with in-situ pollen, Saururus tuckerae S.Y.Sm. & Stockey from Eocene of North America with strong affinities to extant species of Saururus. Recent dated trees suggest, however, an Eocene or younger crown age for the family. METHODS: Dispersed fossil pollen grains from the Campanian (82-81 Ma) of North America are compared to dispersed pollen grains from the Eocene strata containing S. tuckerae, the Miocene of Europe, and extant members of the family using combined LM and SEM imaging. RESULTS: The unambiguous fossil record of the Saururaceae is pushed back into the Campanian (82-81 Ma). Comparison with re-investigated pollen from the Eocene of North America, the Miocene of Europe, and modern species of the family shows that pollen morphology in Saururaceae is highly conservative, and remained largely unchanged for the last 80 million years. DISCUSSION: Campanian pollen of Saururaceae precludes young (Eocene or younger) estimates for the Saururaceae root and crown age, but is in-line with maximum age scenarios. Saururus-type pollen appear to represent the primitive pollen morphology of the family. Often overlooked because of its small size, dispersed Saururaceae pollen may provide a unique opportunity to map the geographic history of a small but old group of Piperales, and should be searched for in Paleogene and Cretaceous sediment samples.

Taxonomic description of in situ bee pollen from the middle Eocene of Germany.

The middle Eocene Messel and Eckfeld localities are renowned for their excellently preserved faunas and diverse floras. Here we describe for the first time pollen from insect-pollinated plants found in situ on well-preserved ancient bees using light and scanning electron microscopy. There have been 140 pollen types reported from Messel and 162 pollen types from Eckfeld. Here we document 23 pollen types, six from Messel and 18 from Eckfeld (one is shared). The taxa reported here are all pollinated by insects and mostly not recovered in the previously studied dispersed fossil pollen records. Typically, a single or two pollen types are found on each fossil bee specimen, the maximum number of distinct pollen types on a single individual is five. Only five of the 23 pollen types obtained are angiosperms of unknown affinity, the remainder cover a broad taxonomic range of angiosperm trees and include members of several major clades: monocots (1 pollen type), fabids (7), malvids (4), asterids (5) and other core eudicots (1). Seven types each can be assigned to individual genera or infrafamilial clades. Since bees visit only flowers in the relative vicinity of their habitat, the recovered pollen provides a unique insight into the autochthonous palaeo-flora. The coexistence of taxa such as Decodon, Elaeocarpus, Mortoniodendron and other Tilioideae, Mastixoideae, Olax, Pouteria and Nyssa confirms current views that diverse, thermophilic forests thrived at the Messel and Eckfeld localities, probably under a warm subtropical, fully humid climate. Our study calls for increased attention to pollen found in situ on pollen-harvesting insects such as bees, which can provide new insights on insect-pollinated plants and complement even detailed palaeo-palynological knowledge obtained mostly from pollen of wind-pollinated plants in the dispersed pollen record of sediments. In the case of Elaeocarpus, Mortoniodendron, Olax and Pouteria the pollen collected by the middle Eocene bees represent the earliest unambiguous records of their respective genera.

Specialized and Generalized Pollen-Collection Strategies in an Ancient Bee Lineage.

Iconic examples of insect pollination have emphasized narrowly specialized pollinator mutualisms such as figs and fig wasps and yuccas and yucca moths. However, recent attention by pollination ecologists has focused on the broad spectra of pollinated plants by generalist pollinators such as bees. Bees have great impact for formulating hypotheses regarding specialization versus generalization in pollination mutualisms. We report the pollination biology of six northern European species of an extinct tribe of pollen-basket-bearing apine bees, Electrapini, of early-middle Eocene age, examined from two deposits of 48 and 44 million years in age. These bees exhibit a pattern of generalized, incidental pollen occurring randomly on their heads, thoraces, and abdomens, obtained from diverse, nectar-bearing plants. By contrast, a more restricted suite of pollen was acquired for metatibial pollen baskets (corbiculae) of the same bee taxa from a taxonomically much narrower suite of arborescent, evergreen hosts with uniform flower structure. The stereotyped plant sources of the specialist strategy of pollen collection consisted of pentamerous, radially symmetrical flowers with a conspicuous gynoecium surrounded by prominent nectar reward, organized in structurally similar compound inflorescences. Pollen specialization in bees occurs not for efficient pollination but rather in the corbiculate Electrapini as food for bee larvae (brood) and involves packing corbiculae with moistened pollen that rapidly loses viability with age. This specialist strategy was a well-developed preference by the early Eocene, providing a geochronologic midpoint assessment of bee pollen-collection strategies.