Botany
A new species of Athrotaxites (Athrotaxoideae, Cupressaceae) from the Upper Cretaceous Raritan Formation, New Jersey, USA.
Journal: Botany
Manuscript ID cjb-2016-0061.R1
Manuscript Type: Article
Date Submitted by the Author: 19-May-2016
Complete List of Authors: Escapa, Ignacio; CONICET-MEF, Paleobotany Gandolfo, Maria;Draft Cornell University, Department of Plant Biology Crepet, William; Cornell University, Department of Plant Biology Nixon, Kevin; Cornell University, L.H. Bailey Hortorium, Plant Biology Section, School of Integrative Plant Science, Cornell University
Keyword: Cupressaceae, Athrotaxites, Cretaceous, Raritan Formation, New Jersey
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A new species of Athrotaxites (Athrotaxoideae, Cupressaceae) from the Upper Cretaceous Raritan
Formation, New Jersey, USA.
Ignacio H. Escapa, Maria A. Gandolfo, William L. Crepet, and Kevin C. Nixon
I. H. Escapa. CONICET, Museo Paleontológico Egidio Feruglio, Avenida Fontana 140, 9100 Trelew,
Chubut, Argentina.
M. A Gandolfo, W. L. Crepet and K. C. Nixon. L.H. Bailey Hortorium, Plant Biology Section,
School of Integrative Plant Science, Cornell University, Ithaca, New York
Corresponding author: Ignacio Escapa (e mail: [email protected]) Draft
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Abstract. A new species of anatomically preserved Cupressaceae is described from the Upper
Cretaceous Raritan Formation (New Jersey, USA). The fossils are charcolified isolated ovuliferous complexes that were studied by means of a combination of MEB images and Micro CT, allowing the observation of morphological and anatomical characters. Each ovuliferous complex bears 3 4 anatropous winged seeds, disposed in one row on a thin medial part of the adaxial side of the ovuliferous complex. Based on the combination of characters such as ovuliferous complex morphology, arrangement of vascular tissues and resin canals, seed number and their morphology, orientation and disposition, these fossils are placed within a new species of the fossil genus
Athrotaxites . The developmental stage of the specimens is analyzed base on comparisons with living representatives of the subfamily Athrotaxoideae (i.e., Athrotaxis spp.), which supports a post pollination stage for these fossils. In addition, the new species is compared with other extant and extinct representatives of basal cupressaceous subfamilies. This new record from the Upper
Cretaceous sediments of New Jersey furtherDraft supports a wider distribution of the subfamily
Athrotaxoideae during the middle part of the Mesozoic, as it has been also noted for other basal representatives of the family Cupressaceae.
Key words: Cupressaceae. Athrotaxoideae. Athrotaxites . Cretaceous. Raritan Formation. New Jersey
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Introduction
The well-preserved, charcoalified angiosperm remains from the Turonian Raritan Formation are
comprehensively known through numerous studies during the last two decades (e.g., Crepet et al. 1992,
2005, 2013; Herendeen et al. 1993, 1994; Nixon and Crepet 1993; Crepet and Nixon 1994; Gandolfo
et al. 1998 a, 1998 b, 2002, 2004; Hermsen et al. 2003; Martínez Millán et al. 2009). Fossil diversity at
the Old Crossman Clay Pit locality (Sayreville, New Jersey) also includes ferns (Gandolfo et al. 1997,
2000) and gymnosperms (e.g. Miller 1985), that, in contrast to angiosperm fossils, have been scarcely
studied. Three dimensionally preserved gymnosperms from this formation have been known since the
beginning of the 20 th century; however, only two species of pinaceous leaves ( Prepinus
crossmanensis and P. quinquefolia ) and the pollen cone Amboystrobus cretacicum (Gandolfo et al.
2001) have been described in detail on the basis of charcoalified specimens.
The conifer family Cupressaceae sensu lato (including the paraphyletic former Taxodiaceae)
has been proposed based on numerous morphologicalDraft and molecular phylogenetic analyses
(Eckenwalder 1976; Hart 1987; Price and Lowenstein 1989; Chase et al . 1993; Brunsfeld et al. 1994;
Stefanovic et al. 1998; Gadek et al. 2000; Kusumi et al. 2000; Farjon 2005). Twenty genera and 142
extant species are recognized in Cupressaceae, which is the most diverse conifer family in terms of
genera. Seven extant subfamilies are currently recognized (Gadek et al. 2000). Interestingly, there is a
strong distinction between the Northern and Southern Hemisphere lineages of Cupressaceae sensu
lato (see Leslie et al. 2012) . Nevertheless, this distinction is less definite if fossil and extant species
are simultaneously considered. For instance, the contrast between the restricted distribution of extant
Cunninghamia (Liu 1966) and the widespread occurrence and notable diversity of the subfamily
Cunninghamioideae during the Mesozoic and Cenozoic has been recognized (Stockey et al. 2005;
Escapa et al. 2008; Serbet et al. 2013; Shi et al. 2014). The extremely low extant diversity and a
similar distribution pattern is also shared with the subfamilies Taiwanioideae and Athrotaxoideae,
which occur in a pectinate arrangement together with Cunninghmamia at the base of the Cupressaceae
phylogenetic tree (Leslie et al. 2012) .
Extant representatives of Cupressaceae subfamily Athrotaxoideae comprise three species:
Athrotaxis selaginoides, A. laxifolia, and A. cupressoides ; all of which are distributed in temperate
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rainforests and alpine vegetation in Tasmania (Cullen and Kirkpatrick 1988; Farjon 2005). However,
A. laxifolia is now considered to be an interspecific hybrid between the other two species, based on morphological (Isoda et al. 2000) and genetic evidence (Worth et al. 2016). Molecular models suggested a Neogene Paleogene age of divergence for the Ahtrotaxis crown group, while the stem age has been estimated as Jurassic Late Cretaceous (Leslie et al. 2012; Mao et al. 2012; Yang et al. 2012).
The fossil record of Athrotaxoideae has been extended to the Early Cretaceous, either with fossils assigned to Athrotaxis or to the fossil genus Athrotaxites (Archangelsky 1963; Miller and LaPasha
1983; Stockey et al. 2005). Fossil evidence for this group includes a small number of Cretaceous records in both hemispheres, together with a more diverse Cenozoic record (see Hill et al. 1993; Hill and Brodribb 1999).
In this contribution, we report the occurrence of athrotaxoid fossils from the Upper
Cretaceous Raritan Formation (New Jersey, USA) based on the presence of charcoalified ovuliferous complexes that belong to the subfamily AthrotaxoideDraftae . The combined use of Scanning Electron
Microscopy (SEM) and Micro CT Scan allowed the observation of detailed anatomical and morphological features supporting the taxonomic placement of these fossils within the fossil genus
Athrotaxites and the erection of a new species, probably representing a stem component within the subfamily Athrotaxoideae.
Materials and methods
Geological setting and paleontological background
The specimens described in this study were collected from the Old Crossman Clay Pit locality, Sayreville (New Jersey, USA), in outcrops of the South Amboy Fire Clay, a member of the
Raritan Formation. The Raritan is part of fluvial sediments composing the Atlantic Coastal plains
(see Petters 1976). Based on regional lithostratigraphy, the sediments of the Raritan Fm. exposed in
New Jersey are divided into five members: (i) the Raritan Fire Clay, (ii) Farrington Sand, (iii)
Woodbridge Clay, (iv) Sayreville Sand, and (v) South Amboy Fire Clay. The age of this member is calculated as Turonian (Upper Cretaceous, 90–94 MY, see Harland et al. 1989) based on stratigraphic and palynological data. Since end of the 19 th century, the Raritan Fm. is considered of special interest
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because of its paleobotanical and palynological content (e.g. Newberry 1895). Subsequent studies led
to the first compendium of the flora by Berry (1911) and later revisions (e.g., Dorf 1952). Notably,
during the last 25 years, the majority of the paleobotanical studies on its paleoflora were focused on
its charcoalified specimens which are highly diverse. The flora is dominated by angiosperms, while
several conifers seem to be well represented but not as abundant; the pteridophytes also are
represented by a few taxa. The angiosperms are represented by flowers, fruits, and seeds of various
phylogenetically disparate groups of magnoliid, monocots and eudicots (e.g., Crepet et al. 1992, 2005;
Herendeen et al. 1993, 1994; Nixon and Crepet 1993; Crepet and Nixon 1994, 1998; Gandolfo et al.
1998 a, 1998b, 2001, 2002, 2004; Crepet 2000; Zhou et al. 2001; Hermsen et al. 2003; Martínez
Millán et al. 2009; Crepet et al. 2013).
Study Technique
Fossil specimens are charcoalified, with three dimensional morphology and excellent
anatomical details. External features wereDraft studied using scanning electron microscopy (SEM).
Specimens were mounted on stubs and sputter coated with gold/palladium in preparation for
examination with a Hitachi 4500 SEM.
In addition, the internal anatomy of one specimen was imaged using a Micro CT (Xradia
model xrm 500) at Cornell University (http://www.ct.cornell.edu/cct/Cornell_CT.html), which
provided serial sections of the fossil that are the bases for the three dimensional models (OsiriX 64 bit
DICOM viewer). The use of this technique for the anatomical study of fossils has been increasingly
used during recent years for fossil vertebrates (e.g., Cunningham et al. 2014), invertebrates (e.g.,
Pakhnevich 2010) and plants (e.g., Crepet et al. 2013; Gee et al. 2014; Steart et al. 2014). This method
has the clear advantage of being non destructive for the fossil specimens under study, which is
particularly relevant when the specimens are rare or unique fossils.
All fossils are deposited in the Cornell University Paleobotany Collection, Plant Biology
Section, School of Integrative Plant Science, Cornell University (Hereafter CUPC).
Terminology. One important argument regarding the interpretation of structures, homologies, and
terminology in extant and fossil conifers is focused on the nature of their reproductive organs. From
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the extensive work of Florin (1938–1945, 1951, 1963), a general consensus was developed in which extant conifer pollen cones are considered as “simple”, while ovulate cones are regarded as typically
“compound” (i.e., composed of two branch orders). However, the universality of these concepts has been widely discussed (e.g., Archangelsky and Cúneo 1987; Hernandez Castillo et al. 2001; Escapa et al. 2008). In terms of ovulate cones, many researchers use the term “bract/scale complex” for describing each unit of the ovuliferous cone, with the scale representing a reduction of a primitive dwarf shoot. In particular, for the family Cupressaceae, some studies have suggested the presence of genera with simple cones (e.g., Libocedrus ), and posited that this could be the ancestral condition for the family (Tomlinson et al. 1993; Tomlinson and Takaso 2002). This interpretation was subsequently discussed by Rothwell et al. (2010) based on a fossil transformational series, documenting the process of reduction from a compound cone to an apparently simple cone. Nevertheless, we prefer the use of descriptive terminology that does not assume homology correspondences for structures that cannot be observed in the specimens being described.Draft
In this context, for seed bearing structures without clear differentiation of bract and scale, the use of “bract scale complex” as a descriptive term implies a series of strong homology hypotheses: (i) the cone is compound, (ii) each unit is composed by a bract and an scale, and (iii) the bract and the scale are fully fused. Because the fossils here described are isolated “scales”, we have decided to use the term “ovuliferous complex”, as it is a more general term and refers to every ovule bearing structure of an ovulate cone (see Escapa et al. 2008). The terms ovuliferous scale (or just scale) and bract are used when an actual division is clearly seen in the ovuliferous complex between a subtending bract and an adaxial ovuliferous scale.
Results
Systematics
Family Cupressaceae
Subfamily Athrotaxoideae
Genus Athrotaxites Unger, 1849
TYPE SPECIES: Athrotaxites lycopodioides Unger
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Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov.
DIAGNOSIS: Isolated ovuliferous complex, clavate spathulate, and rhomboidal in face view. Each
ovuliferous complex with 2 thickened portions, flanking a thinner medial region. Vascular tissue
forming two rows of isolated traces; adaxial vascular traces supplying the seeds; abaxial row with 5
vascular traces, proximally accompanied by resin canals. A single vascular trace extends to the apex
of the complex. 3 4 seeds anatropous inserted adaxially, forming a triangular arrangement. Seeds
cylindrical, with 2 small lateral, integumentary wings.
HOLOTYPE: CUPC 1576
LOCALITY: Old Crossman Clay Pit locality (Sayreville, New Jersey, USA), Raritan Formation (Late
Cretaceous). Draft
PARATYPES: CUPC 1577 1578
ETYMOLOGY: Specific epithet dedicated in honor of Dr. Ruth A. Stockey for her many important
and influential contributions to the understanding of fossil and living plants in general, and conifers in
particular.
DESCRIPTION
Isolated ovuliferous complexes. The description is based on three isolated, charcoalified
clavate spathulate ovuliferous complexes (Figs. 1 4). All the specimens are immature, most likely in a
stage of development just after pollination (see Discussion section), with minor differences in
maturity among them. In all cases the ovuliferous complexes (OCs) are broken at the point of
insertion to the cone axis, showing a rhomboidal scar ca. 0.3 mm in height (Figs 1.1, 1.4, 2.1) that
allows the visualization of the vascular tissues and their largely parenchymatous cortex (Figs. 3 4).
The parenchyma cells are thin walled, polygonal in transverse section, ranging from 6 to 40 µm in
diameter (Figs. 3 4). In front view, the head of the OCs is rhomboidal, up to 0.8 mm in height and
width (Figs. 1 2). The thickness varies along the OC, with a thick basal area (expanded abaxially) up
to 0.4 mm, followed by a thinner area (ca. 0.2 mm) where the ovules are inserted (Figs 3.1, 4.2, 4.3).
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Distal to the ovules insertion point towards the apex, the OC is expanded in thickness (adaxially), up to 0.3 mm, being gradually reduced toward the apex, which is obtuse (Figs 3.1, 4.2, 4.3).
Five vascular traces are seen in the proximal (broken) end of the OC, with diameters varying from 20 µm to 40 µm (Figs. 3.5, 3.6, 4.2, 4.6), the vascular trace in the center is slightly bigger in diameter than the lateral ones (Fig. 4.2). The vascular traces are aligned, on a slightly concave plane, following the morphology of the OC (Fig. 4.2). Basally, the vascular traces are closer to the abaxial side of the OC. At the level of seed insertion, however, they are already close to the adaxial side, and they maintain this position in their course toward the apex of the OC. A second row of much smaller vascular traces positioned adaxially in relationship to the previously described is observed; these traces enter the chalazal end of the seeds and continue irrigating them (Fig. 4.4).
A series of aligned resin canals are seen at the proximal (broken) end of the OC (Fig. 4.6).
Resin canals have prominent epithelial lining. They are generally circular in cross section, with diameters varying from 20 to 70 µm. Proximally,Draft they are oriented parallel to the long axes of the seeds, and turning upwards while following the course of the vascular traces to the apex of the OC.
Approximately ten resin canals are visible at the base of the OC (Fig. 4.6), but they are rapidly reduced in number. There are only five canals at the middle region of the OC (Fig. 4.2), which roughly correspond in number and position to the vascular traces. A single resin canal is present at the level of the seeds, which disappears past the seed insertion area.
All the specimens show three adaxial seeds, inserted closely at the base of OC´s head, in a depression that encloses about half of the seeds’ length (Figs. 1.1, 1.3, 1.4, 1.6). The seeds are arranged in a triangular pattern, with two seeds at the bottom touching each other laterally, and the third one between and above the other two. In addition to the three seeds that are present, one specimen (CUPC 1578) shows a rounded scar that may corresponds either to a seed that has been dispersed or an aborted ovule (Fig. 2.3 at arrow). The seeds are inverted, with the micropyle pointing in the same direction as the broken stalk of the OC (i.e., toward the cone axis. Figs., 1.1, 1.4, 4.3, 4.2).
The seeds are cylindrical to clavate, 0.16 to 0.2 long and 0.14 0.15 mm in diameter medially. Two short (immature) wings protrude from the lateral margins of the seeds (Figs. 1.4, 5.2, 5.3). The wings seem to be a continuation of the seed coat (Figs. 5.2, 5.3). Integument thickness varies among
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different regions of the seed. On its proximal and medial parts, the thickness of the integument varies
between 20 and 35 µm. The thin walled endotesta is often separated from other integument layers,
while the sarcotesta and sclerotesta are usually fused and not distinguishable. Close to the micropyle,
the integument is thickened, and it is formed by a zone of cells that are elongated perpendicular to the
main axis of the seed (Fig. 6.4). These micropyle closing tissues have a thickness of 50 60 µm in
average (Fig. 6.4). The nucellus is contracted to the chalaza (Figs. 3.4, 4.3, 4.4, 5.2, 5.3), and internal
tissues are not differentiable.
Discussion
Systematic affinities
The relationship between completeness and informative value is an important issue when
dealing with assigning fossil taxa to particular lineages or clades. The resolution of this question
seems to be particular to specific clades,Draft since the preservation of the same organs of different groups
may lead to varying degrees of taxonomic precision. To further complicate this scenario, the
informative value of different isolated organs also varies as a direct function of the age. In the case of
Mesozoic conifers, ovulate cones and isolated ovuliferous complexes are frequently assumed to be the
most informative structures for confidently assigning a fossil species to a family or genus, particularly
in the absence of additional preserved organs (e.g., Miller 1982, Rothwell et al. 2012).
Athrotaxites stockeyi is represented by isolated ovuliferous complexes, which bear three ( 4)
adaxially attached seeds with wings of integumentary tissue origination (Fig. 1, Fig. 5). In the context
of modern and fossil post Triassic conifers, the presence of three or more seeds helps to eliminate the
possibility of affinities with several lineages (see Miller 1977, 1982, 1988). The extinct
Cheirolepidaceae always have one to two seeds per ovuliferous complex (e.g., Axsmith et al. 2004;
Escapa et al. 2012, 2013; Stockey and Rothwell 2013), while Pinaceae always have a consistent
number of two seeds per ovuliferous complex (e.g., Smith and Stockey 2001; Klymiuk and Stockey
2012; Rothwell et al. 2012). In the families Araucariaceae, Podocarpaceae, and Phyllocladaceae,
ovuliferous complexes carry a single seed (e.g., Farjon 2010; Stockey 1975). Finally, Taxaceae and
Cephalotaxaceae have 1 2 erect ovules attached in the axil of a vegetative leaf (see Miller 1988 and
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citations therein). Additional differences between A. stockeyi and the aforementioned families exist in the position of the seeds and in their orientation, the presence and origin of seed wings, the presence or absence of enclosing tissues (absent in A. stockeyi ), and the morphology and degree of fusion of bracts and scales. Despite other minor, and less well understood, Mesozoic members of the conifer clade (e.g., Palissyaceae, which also have numerous differences with our specimens; Parris et al.
1995; Pattemore et al. 2014), the combination of characters in A. stockeyi limits its potential affinities to the order Cupressales ( sensu Christenhusz et al. 2011), which includes extant Sciadopitys and
Cupressaceae sensu lato . Furthermore, the presence of 3 (4) seeds with two lateral integumentary wings that are medially arranged on the ovuliferous complex, together with the presence of a thick, clavate spathulate ovuliferous complex, helps to eliminate affinities with Sciadopitys and genera related to New and Old World Cupressaceae (i.e., Cupressaceae sensu stricto . See Schulz et al. 2005).
Basal cupressaceous lineages include five traditional subfamilies: Cunninghamioideae,
Taiwanioideae, Athrotaxoideae, Sequoioideae,Draft and Taxodioideae (Farjon 2005), that form a paraphyletic grade within the family largely supported by both morphological and molecular evidence
(Chase et al. 1993; Brunsfeld et al. 1994; Stefanovic et al. 1998; Kusumi et al. 2000; Hart 1987;
Miller 1988; Mao et al. 2012). The first two subfamilies, which have also been considered as possibly forming a monophyletic group (Farjon 2005; Escapa et al. 2008), are easily distinguished from the fossils described herein because they have thin, coriaceous ovuliferous complexes. Athrotaxites stockeyi also differs from Taxodioideae genera (i.e., Cryptomeria , Taxodium and Glyptostrobus ). All three genera, including extant and fossil species, produce distally toothed ovuliferous scales, which are fairly small relative to the subtending bract at time of fertilization (Takaso and Tomlinson 1990;
Farjon 2005). Furthermore, the ovules are erect and occur in the axil of the ovuliferous scale in all three genera (Takaso and Tomlinson 1990; Farjon 2005; Schulz and Stutzel 2007). Cryptomeria japonica has characteristic ovuliferous scales that are distally toothed. This character is absent in our specimens but is present, with some variations, in fossil species related to this genus (e.g., Kilpper
1968). Modern Glyptostrobus species produce obovate cuneate ovuliferous complexes bearing two seeds, each with a single wing; while the fossil representatives show similarly organized ovuliferous complexes, with wedge shaped scales, proximally fused to the bract but distally free (e.g., Matsumoto
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et al. 1997; Aulenback and LePage 1998). In contrast, A. stockeyi has three to four seeds with two
wings each. Fossil and extant Taxodium spp . have globose cones, which consists of peltate, thick
ovuliferous complexes which are the result of the fusion of a bract to a distally lobed ovuliferous scale
(e.g., Kunzmann et al., 2009), and therefore are clearly different from A. stockeyi . The subfamily
Sequoioideae includes three living genera (Sequoia , Metasequoia, and Sequoiadendron ), with
morphological differences with respect to the species here described such as the peltate head of the
OC in mature cones (i.e., the distal tip of the OC is located centrally on the expanded head), and the
number of seeds (5 10) per OC (e.g., Chaney 1950; Kunzmann and Mai 2011).
Based on similarities in the anatomy and morphology of the ovuliferous complexes and the
ovules/seeds, Athrotaxites stockeyi is most closely comparable to the subfamily Athrotaxoideae,
which has three genera, the extant genus Athrotaxis , and two fossil genera, Athrotaxites and
Athrotaxopsis (Dong et al. 2014). Athrotaxis comprises two to three species confined to the temperate
rain forests of Tasmania (Worth et al. 2016).Draft In modern Athrotaxis , the ovuliferous complex head is
clavate spathulate with an obtuse apex, and ovules are disposed in a single row on a narrowed part of
the OC (see Farjon 2005, Jagel 2001). Seed number and seed morphology in Athrotaxites stockeyi are
within the range of variability shown by extant representatives of the clade (i.e., 3 6).
The use of Micro CT technology has facilitated the observation of anatomical details in the
fossil species, increasing the number of similarities with respect to the athrotaxoid type (Figs. 3 5).
The distribution of the vascular bundles through the different levels of the OC is variable among the
extant species of Athrotaxis (Eames 1913; Hirmer 1936). Nevertheless, they can be characterized by
the presence of a single vascular bundle leaving the cone axis (Fig. 6), two rows of vascular traces in
the middle part of the complex (Fig. 6), which can be reduced to a single row distally (beyond the
level of the seeds). Among extant species this pattern has been described for A. selaginoides and A.
laxifolia (Eames 1913), and the same arrangement is present in A. stockeyi (Fig. 4). On the other hand,
A. cupressoides shows two rows of vascular traces that are extended to the apex of the ovuliferous
complex (Eames 1936). Assuming a compound origin for the Athrotaxis cone, the adaxial row of
vascular traces has been considered to be the vascularization of the ovuliferous scale, while the
abaxial row would correspond to the bract (see Schulz and Stützel 2007). Although the adaxial
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vascular traces of A. stockeyi are smaller than the abaxial ones, and are difficult to distinguish even with the resolution of the Micro CT, it is possible to observe a single trace vascularizing each ovule.
Resin canals accompany both rows of vascular traces in modern species of Athrotaxis (Fig. 6).
A prominent system of resin canals is disposed below the abaxial row of vascular traces, a feature that is comparable to those observed in A. stockeyi (Fig. 4). Also, micro CT data from Athrotaxis selaginoides reveals that less conspicuous resin canals occur close to the adaxial row of vascular traces approximately at the level of the ovule/seed insertion (Fig. 6).
The fossil record of taxodiaceous Cupressaceae also includes numerous genera represented by anatomically preserved seed cones (e.g., Miller 1975; LaPasha and Miller 1991; Nishida et al., 1992;
Nishida et al. 1991; Saiki and Kimura, 1993; Ohsawa, 1994; Yao et al. 1998; Rothwell et al. 2010;
Atkinson et al. 2014; Klymiuk et al. 2015), for which the phylogenetic relationships are currently unknown or poorly understood. Based on characters from the general morphology of the ovuliferous complex, which are highly variable in theDraft context of the family, Athrotaxites stockeyi is comparable with genera such as Cunninghamiostrobus , Sphenolepis (in part), Parataiwania and Hughmillerites , all of which are also characterized by non peltate ovuliferous complexes with tapered apices (see
Atkinson 2014 a and Klymiuk et al. 2015 for recent reviews on basal cupressaceous fossil diversity).
Characters such as organization within the seed cone and vascular tissues at origin are not preserved in Athrotaxis stockeyi, and therefore comparisons are restricted to characters such as the number, position and distribution of vascular traces and resin canals, together with the number, disposition, orientation and morphology of ovule/seeds. Following these features, Athrotaxites stockeyi is comparable with Parataiwania nihongii , from the Upper Cretaceous of Hokkaido, Japan (Nishida et al. 1992). Both species shows a similar arrangement of vasculature and resin canals . In addition,
Nishida et al. (1992) pointed out that Parataiwania , Taiwania , and Athrotaxis are also similar in terms of the parenchymatous composition of the ovuliferous complexes, which is also clear in the specimens of Athrotaxites stockeyi . The absence of an adaxial free tip of the ovuliferous scale (i.e., ligule like scale sensu Nishida et al. 1992) represents the main morphological difference between A. stockeyi and
Parataiwania. The presence of one or several free scale tips also represents a clear difference among our specimens and other basal cupressaceous fossil genera (see Atkinson et al. 2014 a, Table 1), such
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as Hughmemillerites (Rothwell et al., 2011; Atkinson et al. 2014 a), Hubbardiostrobus (Atkinson et al.
2014 b) and most species in Cunninghamiostrobus (e.g., Miller and Crabtree 1989).
Cunninghamiostrobus yubariensis , from the Late Cretaceous of Japan, is also characterized by the
lack of free scale tips on the adaxial side of the ovuliferous complexes (Ohana and Kimura,
1995). However, its ovuliferous complexes are flattened and remain relatively perpendicular to the
axis for its entire length, while in A. stockeyi the distal portion turns distinctly upward. The Jurassic
species Sphenolepis kurriana (see Harris, 1953) shares with Athrotaxites stockeyi similarities in the
arrangement of vasculature and resin canals, and the lack of a scale associated with the seeds (ligule
like scale, sensu Nishida et al. 1992). However, seeds in S. kurriana are disposed in two distinct rows
(Harris, 1953), while Athrotaxites stockeyi and Athrotaxis spp. are characterized by a single, often
curved, row of seeds. Interestingly, other species Sphenolepis , such as S. pecinovensis from the
Cretaceceous of Central Europe possess ovuliferous complexes morphologically comparable with the
ones described here, although its anatomicalDraft features are unknown (Kvacek 1997) and the associated
seeds are characterized by the lack of wings.
Altogether, both morphological and anatomical features of extant species and A. stockeyi ’s
ovuliferous complexes and seeds suggest a close affinity of the specimens described here to members
of Cupressaceae, and with the subfamily Athrotaxoideae. Considering the fossil and the extant
diversity within the group, most features suggest a closer affinity of our specimens with the extant
genus Athrotaxis, the single extant Athrotaxoideae. Comparisons among modern Athrotaxis species
and A. stockeyi suggest closer similarities to A. laxifolia and A. selaginoides than to A. cupressoides.
However, since the morphology of the ovuliferous complexes in extant Athrotaxis largely changes
during development (Jagel 2001), it is crucial to determinate the ontogenetic stage of the ovuliferous
complexes of Athrotaxites stockeyi .
Developmental stage
Seed cones in Cupressaceae are characterized by the morphological variability during
development (e.g., Tomlinson and Takaso 2002), and therefore, determination of ontogenetic stage is
crucial when a fossil species is described . Athrotaxites stockeyi shows numerous features supporting
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its preservation in an advanced stage of development, most likely after pollination. At pollination stages, ovules in Athrotaxis are inserted on a small swelling (Jagel 2001; Jagel and Dörken 2014), which has been interpreted as being part of the ovuliferous scale (Schulz and Stützel 2007). After pollination, this swelling disappears and a new, larger swelling area is produced above the plane of ovule attachment (Jagel 2001), which is interpreted as being part of the bract and its function is related to the closing the cone. This last swelling is already developed in all A. stockeyi specimens, supporting the interpretation that the ovuliferous complexes were in a post pollination stage when the charcoalification process occurred.
Seeds of A. stockeyi have a thick layer of tissue closing the micropyle that we interpret as a homologous tissue to that reported for A. selaginoides (Saxton and Doyle 1929). Particular planes in the tomography series allowed the observation of the cells confirming this closing tissue is composed of a layer of cells that are perpendicular to the long axis of the seed (Fig. 5) in concordance with the tissues detected in A. selaginoides (SaxtonDraft and Doyle 1929).
Finally, several pollen grains of possible cupressaceous affinities have been observed on the adaxial side of the ovuliferous complex of A. stockeyi near the seeds, (Fig. 7). These pollen grains have granulate rugulate exine sculpture; they are approximately 4.5 m in diameter. The grains have general features characteristic of the pollen produced by basal cupressoids, although they are probably extremely reduced in size due to the charcoalification process (see below).
Athrotaxis, Athrotaxites or Athrotaxopsis?
Depending on the author, the fossil record of the subfamily Athrotaxoideae can be considered either as broadly distributed and diverse, or as confined to a few species that are mainly restricted to the Southern Hemisphere. The fossil record of this subfamily goes back the Early Cretaceous, and numerous fossil species have been related to the clade (e.g., Archangelsky 1963; Townrow 1965; Hill and Macphail 1985; Hill et al. 1993; Del Fueyo et al. 2008; Miller and Hickey 2010). However, many of these records have been rejected because the preserved features were insufficient for supporting a definitive assignment to the subfamily. For instance, Florin (1960) in his review of Northern
Hemisphere Athrotaxoideae, rejected all the records reported at that time. Subsequently, Miller and
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Lapasha (1983) comprehensively reviewed in detailed Athrotaxis berryi , a species with broad
distribution in the Northern Hemisphere during the Early Cretaceous (Dong et al. 2014 and citations
therein). In contrast to Florin´s observations, Miller and LaPasha (1983) concluded that the
athrotaxoids were indeed present in the Northern Hemisphere during the Mesozoic.
Traditionally, Mesozoic and Cenozoic species with athrotaxoid affinities have been assigned
either to the extant Athrotaxis (e.g, Archangelsky 1963), or to two fossil genera: Athrotaxopsis
Fontaine (1889), originally defined based on vegetative and reproductive remains collected at the
Potomac Formation (USA), and Athrotaxites Unger (1849), which includes Jurassic vegetative and
reproductive remains with athrotaxoid affinities (Srinivasan 1995). In the past, both fossil genera have
been used randomly to include fossils with Athrotaxoideae affinities, but these lack sufficient
preserved features to be confidently placed within Athrotaxis.
From a taxonomic point of view and considering the preserved characters, Miller and Lapasha
(1984) suggested that there were no validDraft reasons for maintaining the two fossil genera ( Athrotaxites
and Athrotaxopsis ) for these fossils. Likewise, Athrotaxites Unger (1849) has taxonomic priority over
Athrotaxopsis Fontaine (1889). Numerous molecular clock studies have estimated the divergence age
of crown Athrotaxis to have occurred in the Neogene (e.g., Leslie et al. 2012; Mao et al. 2012; Yang
et al. 2012), while its stem lineage has been dated as Mesozoic (e.g., Spencer et al. 2015). More
specifically, the stem age has been suggested as Late Jurassic or Early Cretaceous, which is also
concordant with its fossil record (Dong et al. 2014), since the first appearance of the athrotaxoids is in
the Late Jurassic (Florin 1940). However, by the Early Cretaceous, there are numerous occurrences in
both hemispheres (e.g., Archangelsky 1963; Miller and Lapasha 1983; Chen and Deng 1990; Del
Fueyo et al. 2008). The close similarity of living species distributions in comparison with fossil
distributions also suggests that, most likely, the crown group Athrotaxoideae originated during the
Cenozoic, consistent with previous molecular estimates (Mao et al. 2012). In this context, the
Mesozoic species described as belonging to Athrotaxis , Athrotaxites and Athrotaxopsis , and probably
some with Cenozoic records, most likely represent extinct stem representatives of the subfamily
Athrotaxoideae.
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Miller and Hickey (2010) published a phylogenetic analysis for Athrotaxis parvistrobili , a fossil from the Early Cretaceous of Washington State (USA). Based on a morphological matrix that included sixteen characters from both reproductive and vegetative organs, A. parvistrobili was supported as sister to a clade formed by the extant Athrotaxis cupressoides and Athrotaxopsis berryi , while the other two extant species occupy a basal position between the athrotaxoids. Following the results of this analysis, these fossil species would be part of the crown group Athrotaxis , which would be extended at least to the Early Cretaceous. Also, based on the same analysis, they proposed to recombine Athrotaxopsis berryi and to include it in Ahtrotaxis . We agree that, in the context of this small data matrix, the fossil species is more closely related to Athrotaxites cupressoides than to A. laxifolia and A. selaginoides. However, considering the morphological distance of all organs of
Athrotaxis to the basal most genera in the Cupressaceae (i.e., Cunninghamia and Taiwania ), it is not clear which character configuration is plesiomorphic for the lineage and, therefore, the result obtained by Miller and Hickey (2010) may representDraft a rooting bias, similar to what has been seeing for other conifer families (e.g., Araucariaceae. Escapa and Catalano 2013).
In this context, we recommend keeping the name Athrotaxites for fossil species that cannot be strictly related to Athrotaxis, but that show enough characters to be included in the Athrotaxoideae lineage. Internal phylogenetic resolution within this clade will require detailed analyses based on both continuous and discrete features and on a larger taxon sampling, since previous morphological phylogenetic studies for Cupressaceae are too broad for capturing variation within it (e.g., Farjon
2005; Escapa et al. 2008; Rothwell et al. 2011; Shi et al. 2014), or too narrow to certainly avoid rooting biases.
Numerous studies have been dedicated to reviewing and comparing the fossil species related to Athrotaxis (e.g., Hill and Brodribb 1999; Miller and Hickey, 2010), including a recent compendium of fossil diversity and distribution for the group (Dong et al. 2014). Nonetheless, for a number of reasons, it is not possible to establish detailed specific comparisons among A. stockeyi and previously described fossil species within the subfamily. First, the size of the seeds and ovuliferous complexes cannot be used for establishing comparisons. Lupia (1995), by means of an experimental study, suggested that shrinkage associated to the process of charcoalification would be up to 50%. This
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reduction in size is due to a process with several variables, such as the anatomy of the organ and the
duration and temperature of the charcoalification. Furthermore, Gandolfo et al. (2004) used
observations on the pollination biology of a fossil Nymphaeaceae species to suggest that the
percentage of reduction would be even bigger than previously thought. Ratios and proportions among
different structures should not be used in the case of charcoalifications, since as demonstrated mainly
for angiosperm flowers, the shrinkage of different structures is not uniform (Herendeen et al. 1993;
Lupia 1995). If compared with extant Athrotaxis cupressoides in a similar stage of development (Fig.
8), Athrotaxites stockeyi seems to shows a stronger reduction in size of the tissues of the ovuliferous
complex than of the seeds (Fig. 1).
Secondly, most fossil species previously assigned to Athrotaxoideae are represented by
compressions and impressions of different organs for which anatomical details are unknown (e.g.,
Miller and Hickey 2010; Miller and LaPasha 1983). This precludes the possibility of establishing
further comparisons between A. stockeyiDraft and other fossil species related to the clade. Finally, as stated
before, A. stockeyi is represented by ovuliferous complexes that, even when in a post pollination
stage, are still in development. This is particularly relevant in this lineage, especially for characters
such as the morphology of the ovuliferous complex, which is demonstrated to be highly variable in
ontogenetic development (Jagel 2001).
Conclusions
Athrotaxites stockeyi sp. nov. provides further support for the presence of the subfamily
Athrotaxoideae in the Cretaceous of North America, which is based on anatomically preserved
ovuliferous complexes in a post pollination stage of development. This study demonstrates high
utility of Micro CT technology in order to study charcoalified fossils in general, with a non
destructive methodology with high resolution.
The new species is assigned to the genus Athrotaxites, which is here considered to be part of
the stem group of the subfamily Athrotaxoideae, based on a combination of morphological and
anatomical features of seeds and ovuliferous complexes. Athrotaxites stockeyi is the first anatomically
preserved reproductive organ in the fossil record of the Athrotaxoideae, representing an important
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source of phylogenetic characters that are necessary for elucidating trajectories of morphological and anatomical evolution in basal Cupressaceae lineages. This record increases the conifer diversity of
Old Crossman Clay Pit locality, showing the co occurrence of Pinaceae and Cupressaceae.
The occurrence of this species in Upper Cretaceous sediments of New Jersey also represents further support for the extremely wide distribution of the subfamily Athrotaxoideae during the middle part of the Mesozoic (see Dong et al. 2014). This seems to be a common feature for basal cupressaceous lineages, such as the subfamily Cunninghamioideae, for which the extant distribution is highly restricted to the Southern Hemisphere but was broadly distributed in both hemispheres during the Jurassic (see Atkinson et al. 2014 a).
Acknowledgments
The authors thank Mark Riccio and Jennifer Svitko for extraordinary technical assistance during the development of this paper, and Armin JagelDraft for providing SEM picture of Athrotaxis cupressoides .
We also gratefully acknowledge the comments of two anonymous reviewers. This study was supported in part by CONICET and the Fulbright Foundation (I.H.E.).
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Figure captions
Figure 1. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. general view of
ovuliferous complexes. 1-3. Specimen CUPC 1576 (Holotype). 4-5. Specimen 1577. 1,4. View of the
adaxial side of the ovuliferous complex (OC) showing three seeds and broken proximal end. 2,5.
View of the abaxial side of the ovuliferous complex, showing the rhomboidal shape of the OC head.
3,6. Ovuliferous complex in side view showing the swelling part above the seeds level. All scale bars
= 100 µm.
Figure 2. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. general view of ovuliferous complexes. 1-3. Specimen CUPCDraft 1578. 1. View of the adaxial side of the ovuliferous complex showing three seeds and the broken proximal end. 2. Top view of the OC showing position
of seeds. 3. Detail of the three winged seeds, and the scar of the fourth (arrow). All scale bars = 100
µm.
Figure 3. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsiriX 3D reconstruction
of ovuliferous scale and main planes of section showing its well preserved anatomy. Specimen CUPC
1576 (Holotype). 1-3. OsiriX 3D reconstructions in adaxial (1), abaxial (2) and side view (3). 4.
Longitudinal section of the ovuliferous complex showing a central vascular trace and the central seed.
5. Ovuliferous complex in cross section, near to the level of seed attachment. 6. Section showing two
of the seeds longitudinally dissected. Scale bars: 1 3= 200 µm; 4 6 = 100 µm. Legend: s, seed; vt,
vascular trace; rc, resin canal.
Figure 4. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsirirX data showing
anatomical features. Specimen CUPC 1576 (Holotype). Exact plane of section is shown on the 3D
reconstruction at the top right of each section. 1. Oblique section of the OC, showing central and two
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of the lateral vascular traces in the abaxial row (arrows). 2. Transverse to slightly oblique section of the ovuliferous complex at the level of seeds, showing the adaxial row of vascular traces (arrows) and the associated resin canals. 3. Longitudinal sections of the OC at the level of a lateral seed. 4.
Longitudinal section of the OC at the level of a lateral seed, showing small trace of xylem supplying the seed (arrow). 5. Proximal oblique section of OC, showing system resin canals in longitudinal section 6. Oblique section of the OC showing basal resin canals in transverse section. Arrows indicate vascular traces. All scale bars= 100 µm. Legend: rc, resin canal; mi, micropyle; in; integuments; nu, nucellus.
Figure 5. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsirirX data showing seed anatomical characters. Specimen CUPC 1576 (Holotype). 1. Cross section of a seed at the chalazal end, showing fusion of the integuments and the nucellus. 2. Cross section of a seed at a proximal level showing slightly developed integumentaryDraft wings, integuments, and nucellus. 3. Cross section of seed showing apical area of the nucellus and the integuments. Note that the endotesta is separated from the other integumentary layers. 4. Seed in cross section, close to micropylar region showing elongate cells of tissues closing micropyle. All scale bars= 50 µm. Legend: nu, nucellus; en, endotesta; mct, microyle closing tissues; w, seed wing.
Figure 6. OsiriX data showing anatomical details for Athrotaxis laxifolia (mature) . Specimen BH 106
121. 1. Longitudinal section showing vascular tissues extended to the apex of the ovuliferous complex. Section planes for images 2 3 are indicated. 2. Cross section of ovuliferous complex near the seed insertion region, showing the distribution of the vascular bundles and the resin canals. 3.
Cross section of ovuliferos complex below seed level, showing the distribution of resin canal and vascular bundles. Scale bar = 1 mm. Legends: nu, nucellus; rc, resin canal; vt, vascular trace.
Figure 7. Pollen grains associated with Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. Specimen CUPC 1576. (Holotype). 1. Adaxial side of the ovuliferous complex showing pollen grains (arrows) near to the seeds. Scale bar= 30 µm 2. Detail of pollen grains. Scale bar= 5 µm.
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Figure 8. Ovuliferous complex of Athrotaxis cupressoides in adaxial view (from Jagel, 2001), in a
post pollination stage of development. Scale bar= 0.5 mm. (Courtesy of Armin Jagel)
Draft
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Draft
Figure 1. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. general view of ovuliferous complexes. 1 3. Specimen CUPC 1576 (Holotype). 4 5. Specimen 1577. 1,4. View of the adaxial side of the ovuliferous complex (OC) showing three seeds and broken proximal end. 2,5. View of the abaxial side of the ovuliferous complex, showing the rhomboidal shape of the OC head. 3,6. Ovuliferous complex in side view showing the swelling part above the seeds level. All scale bars = 100 µm.
134x99mm (300 x 300 DPI)
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Draft
Figure 2. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. general view of ovuliferous complexes. 1 3. Specimen CUPC 1578. 1. View of the adaxial side of the ovuliferous complex showing t hree seeds and the broken proximal end. 2. Top view of the OC showing position of seeds. 3. Detail of the three winged seeds, and the scar of the fourth (arrow). All scale bars = 100 µm.
79x73mm (300 x 300 DPI)
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Draft
Figure 3. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsiriX 3D reconstruction of ovuliferous scale and main planes of section showing its well preserved anatomy. Specimen CUPC 1576 (Holotype). 1 3. OsiriX 3D reconstructions in adaxial (1), abaxial (2) and side view (3). 4. Longitudinal section of the ovuliferous complex showing a central vascular trace and the central seed. 5. Ovuliferous complex in cross section, near to the level of seed attachment. 6. Section showing two of the seeds longitudinally dissected. All scale bars: 1 3= 200 µm; 4 6 = 100 µm. Legend: s, seed; vt, vascular trace; rc, resin canal.
208x237mm (300 x 300 DPI)
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Draft
Figure 4. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsirirX data showing anatomical features. Specimen CUPC 1576 (Holotype). Exact plane of section is shown on the 3D reconstruction at the top right of each section. 1. Oblique section of the OC, showing central and two of the lateral vascular traces in the abaxial row (arrows). 2. Transverse to slightly oblique section of the ovuliferous complex at the level of seeds, showing the adaxial row of vascular traces (arrows) and the associated resin canals. 3. Longitudinal sections of the OC at the level of a lateral seed. 4. Longitudinal section of the OC at the level of a lateral seed, showing small trace of xylem supplying the seed (arrow). 5. Proximal oblique section of OC, showing system resin canals in longitudinal section 6. Oblique section of the OC showing basal resin canals in transverse section. Arrows indicate vascular traces. All scale bars= 100 µm. Legend: rc, resin canal; mi, micropyle; in; integuments; nu, nucellus.
237x309mm (300 x 300 DPI)
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Draft
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Draft
Figure 5. Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. OsirirX data showing seed anatomical characters. Specimen CUPC 1576 (Holotype). 1. Cross section of a seed at the chalazal end, showing fusion of the integuments and the nucellus. 2. Cross section of a seed at a proximal level showing slightly developed integumentary wings, integuments, and nucellus. 3. Cross section of seed showing apical area of the nucellus and the integuments. Note that the endotesta is separated from the other integumentary layers. 4. Seed in cross section, close to micropylar region showing elongate cells of tissues closing micropyle. All scale bars= 50 µm. Legend: nu, nucellus; en, endotesta; mct, microyle closing tissues; w, seed wing.
96x107mm (300 x 300 DPI)
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Figure 6. OsiriX data showing anatomical details for Athrotaxis laxifolia (mature). Specimen BH 106 121. 1. Longitudinal section showing vascular tissues extended to the apex of the ovuliferous complex. Section planes for images 2-3 are indicated. 2. CrossDraft section of ovuliferous complex near the seed insertion region, showing the distribution of the vascular bundles and the resin canals. 3. Cross section of ovuliferos complex below seed level, showing the distribution of resin canal and vascular bundles. Scale bar = 1 mm. Legends: nu, nucellus; rc, resin canal; vt, vascular trace.
97x52mm (300 x 300 DPI)
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Draft
Figure 7. Pollen grains associated with Athrotaxites stockeyi Escapa, Gandolfo, Crepet et Nixon sp. nov. Specimen CUPC 1576. (Holotype). 1. Adaxial side of the ovuliferous complex showing pollen grains ( arrows) near to the seeds. Scale bar= 30 µm 2. Detail of pollen grains. Scale bar= 5 µm.
98x111mm (300 x 300 DPI)
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Draft
Figure 8. Ovuliferous complex of Athrotaxis cupressoides in adaxial view (from Jagel, 2001), in a post pollination stage of development. Scale bar= 0.5 mm. (Courtesy of Armin Jagel)
99x115mm (300 x 300 DPI)
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