Thecamoebians (Testate Amoebae) Straddling the Permian-Triassic Boundary in the Guryul Ravine Section, India: Evolutionary and Palaeoecological Implications

Exceptionally well-preserved organic remains of thecamoebians (testate amoebae) were preserved in marine sediments that straddle the greatest extinction event in the Phanerozoic: the Permian-Triassic Boundary. Outcrops from the Late Permian Zewan Formation and the Early Triassic Khunamuh Formation are represented by a complete sedimentary sequence at the Guryul Ravine Section in Kashmir, India, which is an archetypal Permian-Triassic boundary sequence [1]. Previous biostratigraphic analysis provides chronological control for the section, and a perspective of faunal turnover in the brachiopods, ammonoids, bivalves, conodonts, gastropods and foraminifera. Thecamoebians were concentrated from bulk sediments using palynological procedures, which isolated the organic constituents of preserved thecamoebian tests. The recovered individuals demonstrate exceptional similarity to the modern thecamoebian families Centropyxidae, Arcellidae, Hyalospheniidae and Trigonopyxidae, however, the vast majority belong to the Centropyxidae. This study further confirms the morphologic stability of the thecamoebian lineages through the Phanerozoic, and most importantly, their apparent little response to an infamous biological crisis in Earth’s history.

Thecamoebians (testate amoebae) are eukaryotic heterotrophic protists that are polyphyletic in origin, based on molecular RNA analysis [37]. In modern environments, they colonize fresh to slightly brackish water aquatic environments (<4 psu) [38] and they form a simple secreted (autogenous) or agglutinated (xenogenous) test (or shell) that can be preserved in the sedimentary record following their death. Owing to their tight ecological zonation with respect to salinity, pH, and moisture content, subfossil thecamoebian assemblages preserved in late Holocene successions are used to reconstruct salinity [39,40] and sea-level variations [41,42], precipitation variability [43] and anthropogenic impacts on coastal and lacustrine environments [44,45]. However, their pre-Quaternary fossil record is sparse, promoting some uncertainty in our understanding on the evolution of important taxonomic lineages, and the group's response to critical environmental perturbations in Earth's history.
This paper presents the first account of the thecamoebians straddling the PT Boundary at the Guryul Ravine PTB Section in India, and the only one record of Late Permian thecamoebians from the Northwest Himalayan region of Jammu and Kashmir. These results provide direct evidence for the successful crossing of the thecamoebian (testate amoebae) group across the PT Boundary extinction event.

Geological Setting
The Guryul Ravine Section lies in the northernmost Indian state of Kashmir [72] (Fig 4), the geology of which has been extensively reviewed by by Nakazawa et al. [27], Kapoor [1] and Tewari et al [36]. During the Late Permian and Early Triassic, the Kashmir region of the Indian sub-continent was located in northern Gondwana, at 35°S palaeolatitude along the southern margin of the Tethys Sea [73,74]. The depositional setting of the Late Permian Zewan Formation was a shallow marine environment with relatively high terrigenous sediment supply, whereas the Early Triassic Khunamuh Formation was deposited during a transgressive episode [74]. Marine sediments of the Guryul Ravine accumulated above pre-existing volcanic rocks [1]. Isotopic evidence of Proemse et al., 2013 [75] indicates relatively oxic conditions in the shallow marine regions of the Northwest margin of Pangea throughout the Late Permian Mass Extinction (LPME). However, the organic carbon flux study of Algeo et al., 2013 [76], and others [74,77] on the PTB sections have suggested that the well-oxygenated conditions were briefly interrupted by periods of anoxia in the Late Permian-Early Triassic shallow marine environments. The Guryul Ravine Section represents a continuous gradational sequence across the PT Boundary, whereas depositional hiatuses present in other Kumaon and Spiti Himalayas sections preclude a continuous record of paleoenvironmental changes during the PT Boundary Event. Our study of organic matter at the Guryul Section [36], reveals the prevalence of amorphous organic matter (AOM), which perhaps suggests a regional prevalence of anoxic conditions. Stratigraphically, both the Zewan and Khunmuh Formations have been further sub-divided into members and units, respectively, based on lithological and paleontological characteristics. The Zewan Formation is divided into four Members (A-D) [1] (Fig 2). Carbonate rocks and sandy shale comprise Member A, Member B is shale with low carbonate content, Member C is thick bedded sandy limestone, sandy shale and muddy sandstone, and Member D is calcareous muddy sandstone. Only Members A, C and D are rich in marine fossils. An abrupt change in sedimentation to intercalated grey to black limestone and black shale demarcates the onset of the Khunamuh Formation. This Formation is divided in to six members E-J (Fig 2), with the lower units in Member E marking the PT Boundary Event. Member E is sub-divided into Units E 1 , E 2 and E 3 . The Unit E1 contains mixed faunal elements of Late Permian and Early Triassic age. The PT Boundary has been placed at the base of the conodont Hindeodus parvus zone at the contact of E 1 and E 2 (Fig 2).
Pioneering faunal work at the Guryul Ravine PTB Section was carried out by [28], who identified four faunal divisions: I to III in the Zewan Formation (Late Permian) and IV in the Unit E1 of the Khunamuh Formation (Early Triassic) (Fig 2). The faunal divisions I and II correspond to the units A and B, respectively, and contain bryozoans, brachiopods and foraminifers. The faunal diversity of division II is less than that of division I. The faunal division III is displayed by the litho units C and D and shows the dominance of gastropods and bivalves over brachiopods. The faunal division IV is displayed by bivalves and ammonoids. Brachiopods, bivalves and conodonts have also been recovered from this faunal division, which provide chronological control for the section (Fig 2).

Material and Methods
This study is a part of Birbal Sahni Institute of Palaeobotany (BSIP) Project Number 2.3 entitled "Megaand microfloristics of the Permo-Carboniferous sediments of Kashmir region: Evolutionary, biostratigraphical, palaeoecological and palaeophytogeographical implications" under Thrust Area 2-"Phanerozoic Terrestrial and Coastal Ecosystems: Biostratigraphical, Palaeoenvironmental, Palaeoecological And Palaeobiogeographical Aspects". All necessary permits were obtained from the Director, BSIP for the field visit and the described study, which comply with all relevant regulations.
A total of nine bulk sediment samples (GR1 to GR9) were collected from C and D members of Zewan Formation and E Member of Khunamuh Formation (Fig 2), which were processedfollowing the palynological procedure used by Kumar et al, [66]. Samples were first treated with 30% hydrochloric acid followed by wet sieving on a 20 μm-mesh to concentrate microfossils, with the recovered residue mounted on slides with canada balsam. Prepared slides were then studied under a high power light Microscope Leitz Laborlux D to study morphological features of the recovered thecamoebian tests.
The absolute abundance of the number of recovered forms of different species has also been studied ( Fig 5). Morphological data of all the examined individuals has been given in tabular form. (Table 1). The slides are deposited in the repository of the Birbal Sahni Institute of Palaeobotany, Lucknow (www.bsip.res.in/Museum.html) vide museum statement no. 1354. The museum accession numbers of the slides are 14869, 14971-14976 and 14881. Existing  [82] were followed for thecamoebian identification.

Discussion
Despite the overall low recovery of fossil thecamoebians from the Guryul Ravine Section, their recovery at such a critical environmental transition has significant taxonomic and ecological implications. Many of the preserved individuals are intact organic linings as a result of the palynological preparation procedures, and like individuals preserved in Mesozoic amber, allows for an examination of the autogenous (or secreted) mucopolysaccharide test structure. In contrast, other fossil thecamoebians that are typically sieved from bulk sediments, preferentially congregate individuals with agglutinated (or xenogenous) test and damaging many of the organic structures. Structures like inner shell lining, apertural bridges, and also test ornamentation (i.e., spines on the fundus) are all evidenced in the collection from the Guryul Ravine Section (Figs 6-8 (Fig 6J). Such test characteristics are easily observed in the modern thecamoebian lineages and yet rarely observed in fossil examples.
The most significant result of this study is providing direct evidence that very common thecamoebian genera successfully crossed the PT Boundary (e.g., Centropyxis and Arcella). Previously, this could be inferred based on their preservation in both Late Paleozoic and Mesozoic amber deposits, but the Guryul Ravine Section provides individuals on other side of the Thecamoebians from PT Boundary, Guryul Ravine, Kashmir, India boundary at the same paleogeographic locale. These observations attest to the resiliency of the thecamoebian group to global climatic events through the Phanerozoic.
An important omission in this collection is the lack of Difflugia preserved in the Guryul Ravine Section. Many specimens comprise the genus Difflugia, which are abundant in late Holocene freshwater environments. It is possible that taphonomic issues prevented their preservation, but this seems unlikely given the preservation of more fragile autogenous Arcella tests. In other fossil thecamoebian collections that have been sieved out of bulk sedimentary samples, individuals of Difflugia have been more common than Centropyxis [46]. It is possible that this generic bias is related to sample processing techniques (sieving vs. chemical palynological processing).
In general, there are very few studies on thecamoebians from deep-time sequences. The earliest record of thecamoebians is likely from Neoproterozoic marine sediments of Chuar group, Grand Canyon, which were originally described as Vase Shaped Microfossils (VSM's) [65]. Since modern thecamoebians are not found in marine settings, this perhaps suggests a marine origin for the group with their ecological shift to lacutrine or brackish environments in the Early to Middle Paleozoic. During the Carboniferous when land plants greatly diversified and formed widespread swamp depositional environments, thecamoebians were preserved in the resultant organic-rich coal deposits now presently located in Nova Scotia, Canada [99,69,56]. The recovered fossil thecamoebians from the PT Boundary at the Guryul Ravine Section can be interpreted in one of two ways. The thecamoebians were transported to the locality along with other terrigenous material from the adjacent coastal zone, or perhaps they represent an in situ or primary assemblage. The implications of the later interpretation would be that some members of the thecamoebian group also occupied marine habitats in some capacity through the entirety of the Paleozoic. For example, the two Late Permian occurrences of Centropyxis, one from the fresh water sediments of the Raniganj Formation, Godavari Graben [68] and the other from the marine sediments of Zewan Formation (this study) suggest that the genus occupied both fresh water as well as marine environments during this time. Similarly, the genus Arcella has been reported from Carboniferous freshwater coals [56] and from the Late Permian sediments of Raniganj Formation at Lingala-Koyagudam coal belt, Godavari graben [68], whereas this study presents the occurrence of Arcella from the marine Early Triassic Unit E3. If indeed thecamoebians occupied marine environments throughout the Paleozoic, however, it remains puzzling why more occurrences of marine thecamoebians have not been reported in the global palynological surveys of Paleozoic marine sediments. Therefore, it remains important to continue documenting fossil thecamoebians to accurately resolve their Paleozoic paleoecological tolerance to salinity and evolutionary history.
Another important observation based on this PT Boundary collection is the variability in the absolute number of spines ornamented onto the tests in the species Centropyxis. The taxonomy of these species remains complex, in part because of the intragradational character in the external morphology of many thecamoebian species [100,101]. Some authors consider dividing this genus into species based on the absolute number of spines ornamenting the test or their geometric orientation [102,103]. Most thecamoebian workers agree with the notion of considerable phentoypic plasticity in the Centropyxis genus, and recognize that the most important variability for paleoecologic work is taxonomic consistency. Here, the recovered centropyxids are also phenotypically diverse, with multiple numbers of spines that we divide into morphotypes, or variants, based on the absolute number of spines. For example, the diagnosis of Centropyxis aculeata var. spinosa includes an individual with >3 spines ornamenting the posterior of the test [103]. The ability to discern such taxonomic detail is remarkable given the age of the specimens, and indicates that phenotypic plasticity in the number of spines is a long-lived characteristic in this genus. Even in modern thecamoebians, however, the functional ecology of this test ornamentation remains unknown.

Conclusions
This study documents exceptionally well-preserved thecamoebians across the PT Boundary event from the Guryul Ravine Section in Kashmir, India. The site was paleogeographically situated at the northern margin of Gondwana, and the recovered thecamoebians are from the Late Permian Zewan and the Early Triassic Khunamuh Formations. The Guryul Ravine Section is an archetypal PT Boundary sequence, and the thecamoebians provide an additional faunal signature to the previously documented foraminifera, brachiopods, bivalves, ammonoids and conodonts. The recovery and preservation of the thecamoebians from the Guryul Ravine Section is not only new to this section but for any PTB section worldwide. The preservation of these fragile microfossils was likely enhanced by punctuated local or regional anoxic/dysoxic conditions, which are evinced by the presence of amorphous organic matter in this section [36]. Even the intraspecific variability within the Centropyxis genera could be observed in the recovered individuals. Thecamoebians have a poorly resolved fossil record, but the results presented here confidently indicate that some of the most common modern thecamoebian genera successfully transitioned across the PT Boundary extinction event with apparently little ecologic challenge.