American Journal of Botany 99(6): 1069–1082. 2012.

A LOWER (VALANGINIAN) CONE PROVIDES THE EARLIEST RECORD FOR P ICEA () 1

A SHLEY A . K LYMIUK 2,3,5 AND R UTH A . S TOCKEY 2,4,6

2 Department of Biological Sciences, CW 405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2E9; 3 Department of Ecology & Evolutionary Biology, Haworth Hall, 1200 Sunnyside Ave, University of Kansas, Lawrence, Kansas 66045-7534 USA; and 4 Department of Botany and Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, Oregon 97331-2902 USA

• Premise of study: Sequence analyses for Pinaceae have suggested that extant genera diverged in the late Mesozoic. While the fossil record indicates that Pinaceae was highly diverse during the Cretaceous, there are few records of living genera. This description of an anatomically preserved seed cone extends the fossil record for Picea A. Dietrich (Pinaceae) by ~75 Ma. • Methods: The specimen was collected from the Apple Bay locality of Vancouver Island (Lower Cretaceous, Valanginian) and is described from anatomical sections prepared using cellulose acetate peels. Cladistic analyses of fossil and extant pinaceous seed cones employed parsimony ratchet searches of an anatomical and morphological matrix. • Key results: This new seed cone has a combination of characters shared only with the Picea A. Dietr. and is thus de- scribed as Picea burtonii Klymiuk et Stockey sp. nov. Bisaccate pollen attributable to Picea is found in the micropyles of several ovules, corroborating the designation of this cone as an early . Cladistic analyses place P. burtonii with extant Picea and an Oligocene representative of the genus. Furthermore, our analyses indicate that Picea is sister to Cathaya Chun et Kuang, and P. burtonii helps to establish a minimum date for this node in hypotheses of phylogeny. • Conclusions: As an early member of the extant genus Picea , this seed cone extends the fossil record of Picea to the Valanginian Stage of the Early Cretaceous, ca. 136 Ma, thereby resolving a ghost lineage predicted by molecular divergence analyses, and offers new insight into the evolution of Pinaceae.

Key words: conifer; Cretaceous; Midoriphyllum ; Picea ; Pinaceae; Pityostrobus ; seed cone.

The family Pinaceae contains 225–232 extant species within 2002 ; Gernandt et al., 2008, 2011 ; Rothwell et al., 2012). 11 genera, ( Farjon, 1998 , 2010, Eckenwalder, 2009 ), many of Divergence estimates indicate long branches at all basal nodes which are biologically and economically important components within Pinaceae, particularly subtending the genera Pinus L., of northern hemisphere forests. The phylogeny and evolution of Picea, and Cathaya (Gernandt et al., 2008), which form the Pinaceae are subjects of intense interest, and our understanding subfamily Pinoideae (Gernandt et al., 2011), to which some au- is rapidly expanding as new data accumulates (e.g., Wang et al., thors also attribute the sister taxa Larix Mill. and 2000; Mathews, 2009; Rudall et al., 2010; Gernandt et al., Carrière, (e.g., Price, 1989; Liston et al., 2003). Analyses using 2011 ). While phylogenetic analyses of nonmolecular ( Hart, a relaxed fossil-calibrated molecular clock (Gernandt et al., 1987 ) and molecular ( Wang et al., 2000 ; Liston et al., 2003 ; 2008 ) suggest these fi ve genera share a minimum divergence in Eckert and Hall, 2006 ; Gernandt et al., 2008 ) characters for the Late or Early Cretaceous. Pinaceae have provided resolution of the relationships between During the late Mesozoic, pinaceous were subject to extant genera, the modern genera have shown themselves to be rapid evolution and radiation, as evidenced by a preponderance relicts of a more diverse evolutionary history ( Miller and Robison, of diverse seed cone morphologies. These include the newly 1975 ; Miller, 1976a , 1977a , 1988 ; Robison and Miller, 1977 ; described Jurassic Eathiestrobus mackenziei Rothwell, Mapes, Alvin, 1988; Miller and Li, 1994; Smith and Stockey, 2001, Stockey et Hilton ( Rothwell et al., 2012 ), the monophyletic genus Pseudoaraucaria Fliche, which contains six species ( Alvin 1957a , 1957b , 1960 , 1988 , Miller and Robison, 1975 ; Miller, 1 Manuscript received 5 December 2011; revision accepted 12 April 2012. 1976a , 1977a ; Smith and Stockey, 2001 , 2002 ), Obirastrobus The authors wish to acknowledge C. N. Miller, Jr., for the University of Ohsawa, Nishida et Nishida (1992), with two species, and an Montana Conifer Reference Collection and A. R. Sweet, Geological Survey assemblage of 27 anatomically distinct cones described as of Canada (Calgary), for palynological assistance. The authors thank Gar Pityostrobus Nathorst emend. Dutt, which has long been recog- Rothwell, and two anonymous reviewers for commentary that greatly nized as polyphyletic ( Miller, 1976a , 1988 ; Stockey, 1981 ; improved the manuscript. This research was supported in part by Natural Falder et al., 1998 , Gernandt et al., 2011 ). That this portion of Sciences and Engineering Research Council of Canada grant A-6908 to the family’s morphological diversity has been lost through ex- R.A.S. and National Science Foundation grant EF-0629819 to Gar W. tinction is a signifi cant hurdle to a complete understanding of Rothwell, Gene Mapes, and R.A.S. and was conducted by A.A.K. in partial the phylogeny and of Pinaceae, to say little of diffi - fulfi llment of degree requirements (M.Sc., University of Alberta, Edmonton, AB, Canada). culties inherent in determining the evolutionary signifi cance of 5 Author for correspondence (e-mail: [email protected]) various anatomical characters of living genera. 6 E-mail: [email protected] Comparative and cladistic attempts to ascertain the evolu- tionary relationships of Cretaceous fossils with respect to ex- doi:10.3732/ajb.1100568 tant genera have produced results that are not always concordant

American Journal of Botany 99(6): 1069–1082, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America 1069 1070 AMERICAN JOURNAL OF BOTANY [Vol. 99

with phylogenies of extant Pinaceae derived from molecular 632, http://www.morphobank.org) includes 54 anatomical and morphological systematics ( Miller 1976a ; Wang et al., 2000 ; Shang et al., characters, as defi ned by Gernandt et al. (2011), who revised and expanded earlier 2001 ; Smith and Stockey, 2001 , 2002 ; Gernandt et al., 2008 , versions of the matrix (Smith and Stockey, 2001, 2002 ). Characters and character state defi nitions employed here follow Gernandt et al. (2011) . The matrix was 2011 ), and few Mesozoic fossils are attributable to living gen- expanded to include the new specimen from Apple Bay, and the Oligocene seed era. Here, we present an anatomically preserved pinaceous seed cone Picea diettertiana Miller (1970), which was used as a comparative standard. cone from the Valanginian (Early Cretaceous) Apple Bay local- In addition, anatomical features of extant Picea were reinvestigated (Morphobank ity of Vancouver Island, British Columbia, Canada. The new P632) using the University of Montana Conifer Reference Collection, now cone is most similar to extant Picea , an assignment that is cor- housed at Oregon State University, Corvallis. As a result of re-examining roborated by the presence of Picea pollen within its ovules. these specimens, we have modifi ed the matrix such that Character 3 (secondary xylem continuity) for the operational taxonomic unit (OTU) Picea is scored as Picea -like , Midoriphyllum piceoides Stockey et Wiebe “0”, or continuous, whereas in previous analyses this character was treated as (2008), that are also present at the locality may represent the polymorphic. vegetation of this early member of the spruce lineage, but de- In all analyses, Cryptomeria japonica (L. f.) D. Don and Sciadopitys verticil- velopment of a whole-plant concept reconciling the two taxa lata (Thunb.) Siebold et Zucc. were defi ned as outgroups, as per Smith and may be precluded by the nature of the depositional setting of the Stockey (2001, 2002 ). For each analysis, 10 sequential “multiratchet” -searches strata at Apple Bay. were performed under the parsimony ratchet perturbation algorithm ( Nixon, 1999), in the program TNT (Goloboff et al., 2008) spawned through the program Winclada (Asado, version 1.1 beta, by K. Nixon, Cornell University). Characters 13, 16, 43, 51, and 54 were ordered, following Gernandt et al. (2011) . Ratchet MATERIALS AND METHODS perturbation sampled 10% of the parsimony-informative characters through 5000 iterations per replication, with three held per iteration. We report Nelson Geologic and depositional setting — An isolated immature seed cone was consensus phylogenies, which are semistrict in that mutually compatible clusters collected at the Apple Bay locality of northern Vancouver Island, (50 ° 36 ′ 21 ″ N, are retained (Nelson, 1979, and see Nixon and Carpenter, 1996); bootstrap values 127 ° 39 ′ 25 ″ W; UTM 9U WG 951068), where sedimentary strata crop out as a ( Felsenstein, 1985 ) were produced from 1000 replicates, with 10 trees held for rocky beach on the north shore of Holberg Inlet. The Apple Bay locality is one each of 100 multiple tree-bisection-reconnection (TBR) search replications. of several sedimentary basins associated with the Wrangellian terrane, the sedi- ments of which were deposited during a continental transgressive sequence, having eroded from the Jurassic Bonanza rhyolites ( Muller et al., 1974 ; Hammack RESULTS et al., 1994). Apple Bay has been interpreted as a Lower Cretaceous Longarm Formation (Fm) equivalent (Jeletzky, 1976; Haggart and Tipper, 1994) and was originally regarded as Barremian because a single type of monocolpate reticu- Systematics— late pollen grain is present within the sediments ( Sweet, 2000 ). However, grains Order—Coniferales of this type extend back to the and Jurassic (Cornet, 1989; Cornet and Family—Pinaceae Lindley Habib, 1992 ), and while they might belong to basal angiosperms, they are also Genus—Picea A. Dietrich attributable to extinct gymnosperm lineages ( Friis et al., 2011 ). Thus, Apple Species—Picea burtonii Klymiuk et Stockey sp. nov . Bay represents a pre-angiosperm fl ora, and recent oxygen isotope analyses in- dicate the locality is ~136 Ma, corresponding to the mid-late Valanginian (D. R. Gröcke, Durham University, personal communication). Specifi c diagnosis — Seed cone, at least 3.2 cm long and 0.5 cm The 6.2 m thick section is composed of 20 beds, of predominantly clast- in diameter, cylindrical. Bract-scale complexes helically arranged. supported cryptically bioturbated (sensu Pemberton et al., 2008 ) sandstone ce- Pith parenchymatous with scattered sclereids. Vascular cylinder mented by CaCO3 . Thirteen beds contain semisideritic concretions within endarch, continuous; 8–10 tracheids wide. Secondary xylem tra- which plant remains are anatomically preserved. Sandy stacked amalgamated cheids with helical and scalariform thickenings. Cortex mostly bedsets, like those at Apple Bay, form on delta-fronts under hyperpycnal condi- parenchymatous with scattered sclerenchyma and 12–15 resin ca- tions ( MacEachern et al., 2005 ). These conditions may be persistent, as in the case of seasonal storms or fl oods, which produce concomitant increases in phy- nals. Vascular traces to ovuliferous scale abaxially concave; terete todetrital infl ux (MacEachern et al., 2005). Stockey et al. (2006) previously bract trace arising separately from vascular cylinder. Bract 0.6– suggested that the strata at Apple Bay are tempestites. The remains of moss and 0.7 mm long, triangular in cross section, with two lateral resin liverwort gametophytes, lycophytes, Equisetum L., and many pteridophytes— canals; separating from ovuliferous scale at lateral margins. Ovu- including sporelings, the dipteridaceous fern Hausmannia morinii Stockey, liferous scale at least 1.13 mm long and borne perpendicular to Rothwell et Little (2006) , and numerous osmundaceous and pteridaceous fertile cone axis; resin canals both abaxial and adaxial to vascular strands remains—all combine to form a cohesive picture of a forest-fl oor fl ora marginal to a river or stream, constituents of which were periodically swept into the throughout and also between vascular strands distal to seed body. marine basin during storm events. two per scale, winged.

Specimen preparation — The concretion (P16098) containing the specimen Holotype hic designatus— Peels and slides of specimen UAPC- was sectioned into ~0.75 cm thick wafers, exposing the cone in oblique longi- ALTA P16098 Bbot, Bxs, and Ctop, housed in the University of tudinal section. Using the cellulose acetate peel technique (Joy et al., 1956), we Alberta Paleobotanical Collection (UAPC-ALTA). prepared consecutive longitudinal sections from both faces, and reoriented one (P16098 Bbot) to obtain serial transverse sections. Peels of the specimen were mounted on glass slides, using xylene-soluble Eukitt (O. Kindler GmbH, Stratigraphy —Longarm Formation equivalent. Freiberg, Germany) mounting medium. Images were captured with a Power- phase (Phase One, Copenhagen, Denmark) digital scanning camera mounted on Age —Valanginian (Early Cretaceous, ~136 Ma). a Leitz Aristophot bellows camera and Zeiss WL compound microscope; a Nikon DS-Ri1 camera mounted on a Nikon Eclipse 80 i compound microscope; Etymology —This seed cone is named for the motion picture and a Microlumina System version 1.2 (Leaf Systems, Westborough, Mas- storyteller Tim Burton. The ovuliferous scale, when viewed in sachusetts, USA). Images were processed with Adobe (San Jose, California, transverse section (see Fig. 2E, 2F ), seems a demonstrable case of USA) Photoshop CS3. “life imitating art”. Phylogenetic analyses — Analyses comparing the new cone to extant and fossil taxa were conducted using a morphological matrix for seed cones of pina- Description — General features— This immature seed cone is ceous affi nity, adapted from previous studies ( Smith and Stockey, 2001 , 2002 ; 3.2 cm long and 0.5 cm wide, and nearly complete (Fig. 1A), Gernandt et al., 2011). The matrix (available online as Morphobank Project although the apex is not preserved, and the exterior bears some June 2012] KLYMIUK AND STOCKEY—EARLIEST FOSSIL RECORD OF PICEA 1071 evidence of abrasion ( Fig. 1A, 1B ). Diagenetic effects, includ- the seed ( Fig. 2F ), before thinning distally to form the wing ing pyritization and calcium recrystallization, have obscured ( Fig. 3A ). Seed wings are 545 µm long and 18 µm thick distal some internal tissues. to the seed (Fig. 3A). The seeds exhibit endotestal, sclerotestal, and sarcotestal integumentary layers ( Figs. 2E, 3A ); some con- Cone axis— The pith, 330–350 µm in diameter, is composed tain nucellar tissue ( Fig. 3A ) attached to the integument ( Figs. of parenchyma and scattered sclereids ( Fig. 1B, 1C ). It is sur- 3A, 3C). Megagametophyte tissue is also present in a few seeds rounded by a continuous endarch vascular cylinder exhibiting (Fig. 3A), as well as bisaccate pollen grains, several of which primary and secondary growth (Fig. 1C), which is occasionally are embedded within shrunken nucellar tissue (Fig. 3C). A interrupted by divergence of bract-scale complexes ( Fig. 1B ). greater number of seeds have pollen grains preserved in their The xylem is 110 µm (8–10 tracheids) wide and represents a micropyles ( Fig. 3B ). single growth increment ( Fig. 1C ). Tracheids, when viewed in longitudinal section (Fig. 2D), exhibit helical to scalariform Pollen— Grains are bisaccate and exhibit distinct irregular secondary wall thickenings. No resin canals are present within verrucate endoreticulations, which become fi ner with proximity the xylem, and phloem is not preserved ( Fig. 1C ). The cortex is to the corpus ( Fig. 3D ). Sacci are 44–48 µm long, and 20–28 µm 135–160 µm wide, and predominantly parenchymatous, with wide. Sacci are borne low with respect to the proximal aspect of scattered sclerenchyma throughout ( Fig. 1C ). A ring of 12–15 the corpus, and their broad attachment forms an obtuse angle resin canals, 65 µm in diameter, occurs medially within the cor- (Fig. 3D, at arrow). The suboblate corpus is 36–42 µm wide, tex ( Fig. 1B, 1C ). 48–52 µm long. The cappa, or proximal surface of the corpus, is psilate to scabrose, but not otherwise ornamented. The capp- Bract-scale complex— The bract-scale complexes are heli- ula, or distal surface, exhibits sculptured ridges that largely cally arranged and borne at right angles to the cone axis (Figs. occlude the germinal furrow ( Fig. 3E , at arrow). 1D, 1E, 2A). At their origin from the vascular cylinder, the vas- cular traces to the ovuliferous scale and bract are entirely sepa- Phylogenetic analysis — Parsimony ratchet analysis of the rate ( Fig. 2B, 2C ). The scale trace is initially derived from two pinaceous seed cone matrix ( Smith and Stockey, 2001 , 2002 ; separate bundles on either side of the gap, which unite within the Gernandt et al., 2011), subsequent to correction to the scoring inner cortex to form a single abaxially concave strand (Fig. 2C). of Character 3 for the OTU Picea , produced a single most par- Within the ovuliferous scale, the vascular tissue rapidly di- simonious tree (L = 256, CI = 25, RI =61) that was topologi- vides, forming multiple strands, no more than 3–5 cells in cally consistent with the tree produced by Gernandt et al. diameter. (2011) . When the matrix was reanalyzed after the addition of The resin canal system of the bract-scale complex is derived the fossil seed cones Picea burtonii and P. diettertiana ( Miller, from perpendicular branching of the axial resin canals within 1970), both fossil clustered with the OTU Picea in the the inner cortex ( Fig. 2A ). The resultant two resin canals ( Fig. 2B ) Nelson consensus of two equally parsimonious trees ( Fig. 4 .; accompany the vascular tissue to the base of the bract-scale L = 262, CI = 0.24, RI = 0.59). Consensus topology was other- complex (Fig. 2C) whereupon they ramify adaxially, producing wise unaltered by the addition of these taxa, and support for a the two resin canals ultimately associated with the interseminal sister-group relationship between Larix and Pseudotsuga was ridge (Fig. 2B, 2E); the two most abaxial canals remain associ- signifi cant (BS = 81). ated with the bract and accompany the terete bract trace into the free part of the bract ( Fig. 2A ). Subsequent branching of the two adaxial canals produces four additional canals abaxial to DISCUSSION the vascular tissue of the ovuliferous scale (Fig. 2B, 2E). At the level of the seed body, these canals undergo lateral divisions Affi nities of the Apple Bay cone — The pinaceous affi nities ( Fig. 2E ), and a further division distal to the seed body produces of Picea burtonii are evident in the helical arrangement of two abaxial series ( Fig. 2F, 2G ). bract-scale complexes, of which the bracts are diminutive. As Morphologically, bracts are 0.6–0.7 mm long and 0.2 mm in all members of Pinaceae, the scales bear two adaxial seeds, wide, triangular in transverse section, and exhibit basal lobes with wings derived from ovuliferous scale tissue. Morphologi- (Fig. 2A, 2E). Separation of the bract from the ovuliferous scale cally, the cone is slender, and cylindrical, which is typical for occurs along the lateral margin. Ovuliferous scales are at least pinaceous cones ( Miller, 1976a ; Farjon, 1998 ). Historically, af- 1.13 mm long and vary in width from 0.760 mm at their base to fi nities of many fossil seed cones were assessed on the basis of 0.67 mm immediately distal to the seed ( Figs. 1D, 2A, 2E, 2F ). such gross morphology alone (Miller, 1974), resulting in the Near the cone axis, the scale is 0.36 mm thick, thinning to 0.2 mm assignment of many Cretaceous cones to Picea (i.e., Berry, distally. The abaxial surface of the scale is sclerotic (Fig. 2E), 1905 ; Penny, 1947 ). However, Miller (1974 , 1976a ) demon- and parenchyma adaxial to the vascular tissue extends up be- strated that these fossils bear little anatomical similarity to ex- tween the paired seeds to form an interseminal ridge ( Fig. 2E, tant Picea ( Miller, 1974 , 1976a ). Most pinaceous seed cones 2F ), 80 µm high and 145 µm wide, that is no more than half the were thereafter assigned to the polyphyletic Pityostrobus height of the seeds (Fig. 2E). Parenchyma of the interseminal assemblage. ridge is continuous with that of the ovuliferous scale, from Cladistic analyses ( Smith and Stockey, 2001 , 2002 ; Gernandt which the seed wings are also derived ( Fig. 2F ). et al., 2011; Fig. 4) have attempted to place fossil seed cones in phylogenetic context, and heuristic (Gernandt et al., 2011) and Seeds— The adaxial surface of each ovuliferous scale bears parsimony ratchet ( Fig. 4 ) searches produce two major clades two winged ovules (Figs. 2A, 2E, 3A), which are inverted with generally corresponding to pinoid and abietoid lineages as de- micropyles (Fig. 3B) facing the cone axis. Seed bodies are 400 µm rived from sequence data. The basic taxonomic division of the long, 90 µm high, 170 µm wide, and are partially enclosed by family into two subfamilies, with extant Cathaya , Picea , and ovuliferous scale tissue, which is thickest at the chalazal end of Pinus comprising Pinoideae, and Abietoideae composed of 1072 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 1. Picea burtonii sp. nov. Holotype UAPC-ALTA P16098. (A) Oblique longitudinal section through ovulate cone, showing helically arranged bract-scales complexes. Bbot #2, scale = 1 mm. (B) Transverse section near base of cone. Note ovuliferous scale bearing two adaxial ovules (at arrows). Bxs #92, scale = 1 mm. (C) Transverse section of cone axis, showing sclerotic pith (p), vascular cylinder, and cortex with resin canals (r). Bxs #80, scale = 0.5 mm. (D) Longitudinal section of cone showing attachment of ovule-bearing bract-scale complexes at right angles to cone axis. Bbot #13, scale = 1 mm. (E) Tangential longitudinal section of cone showing bract attachment at base of bract-scale complexes. Bbot #9, scale = 1 mm. June 2012] KLYMIUK AND STOCKEY—EARLIEST FOSSIL RECORD OF PICEA 1073

Abies Mill., Trew, Carrière, derived and likely represent convergence. Indeed, Abies and Gordon, and Carrière, is well established ( Price et al., some species of Cedrus lack interseminal ridges, as do sev- 1987; Price, 1989; Wang et al., 2000; Liston et al., 2003; Eckert eral crown pinoid fossils, including Pityostrobus bommeri and Hall, 2006; Gernandt et al., 2008). These two lineages have (= P. bernissartensis , Alvin, 1953 , 1957b ), P. cornetii (Coemans) been recovered with UPGMA immunological comparisons Alvin, and P. hautrageanus Alvin + P. macrocephalus Dutt ( Price et al., 1987 ), in Fitch parsimony cladistic analyses utiliz- ( Dutt, 1916 ; Alvin, 1953 , 1960 ; Smith and Stockey, 2001, ing morphological and nonmolecular characters ( Hart, 1987 ), 2002 ). Picea burtonii, in comparison, has interseminal ridges and in analyses of mitochondrial nad5, nuclear 4CL, and that extend no more than half the height of the seed body, a cpDNA rbcL and matK loci ( Wang et al., 2000 ; Gernandt et al., condition that is probably plesiomorphic. 2008 ). Recent analyses of seed cone anatomy ( Gernandt et al., The second putative synapomorphy uniting the abietoids is the 2011 ; Fig. 4 ) have clarifi ed the position of several fossil members presence of integumentary resin vesicles, which Picea burtonii of the family, indicating that Pinus, as presently circumscribed, is lacks. Among extant genera, integumentary resin vesicles are re- paraphyletic with the exclusion of several Pityostrobus taxa. stricted to the subfamily Abietoideae (sensu Gernandt et al., These analyses also indicate that extant genera Larix and Pseudo- 2008 , 2011 ). Among fossil abietoids, resin vesicles occur in four tsuga are basal, and there is support for a sister-taxon relationship pseudoaraucarian species ( Alvin 1957a , 1957b , 1988 ; Miller and between Picea and Cathaya ( Gernandt et al., 2011 ; Fig. 4 ). Robison, 1975 ), Pityostrobus makahensis Crabtree et Miller In our analysis, Picea burtonii clusters with both the OTU (1989) , P. cornetii , and [Pityostrobus oblongus (Carruthers) Picea and an Oligocene cone, P. diettertiana ( Miller, 1970 ), Seward + P. leckenbyi (Carruthers) Seward] ( Creber, 1960 ). thereby providing a minimum age constraint for the Picea- Thus, Picea burtonii is clearly attributable to the pinoid lin- Cathaya node. Although the specimen has been subject to eage, which is dominated by taxa exhibiting affi nities to Pinus taphonomic effects, individual ovuliferous scales exhibit a high (Fig. 4). Close affi nities between P. burtonii and the Pinus level of preservational acuity, permitting us to observe combi- crown group may be dismissed, as P. burtonii does not exhibit nations of characters that are shared only with extant Picea , and characters thought to be synapomorphic for these taxa. In Picea distinguish the new cone from all other Mesozoic taxa. burtonii, the ovuliferous scale thins distally and is therefore un- Picea burtonii exhibits a number of characters that indicate a like that of Pinus, which thickens and supports an umbo, and close affi nity with other pinoid taxa (sensu Gernandt et al., 2011 also dissimilar to the distally thickened condition observed in and Fig. 4), and exclude it from the abietoid lineage. Morpho- many of the Pityostrobus species that cluster with Pinus , in- logically, the distal portion of the scale (beyond the seed body) cluding Pityostrobus andraei (Coemans) Seward, P. argonnen- does not turn sharply upward, as in Cedrus , Abies , Keteleeria , sis (Fliche) Creber, P. jacksonii Creber, P. kayeii Miller and some species of Pseudoaraucaria ( Miller, 1976a ; Smith et Robison, P. lynnii Miller, P. palmeri Miller, and ( P. hautrag- and Stockey 2001, 2002 ). As in most pinaceous conifers, the eanus Alvin + P. macrocephalus Lindley et Hutton). Bract and bract subtending the ovuliferous scale is short, whereas it is scale traces that are united at their origin from the vascular cyl- elongated in Abies , Tsuga , and some species of the basal sister inder occur only in Pinus among living genera (Miller, 1976a; taxa (Larix + Pseudotsuga ) (Silba, 1986; LePage and Basinger, Smith and Stockey 2001 , 2002 ), but the phylogenetic impor- 1995). Similarly, only the extant abietoids Abies , Cedrus , and tance of this character is unclear: Although most non-Pinus Pseudolarix possess ovuliferous scales that abscise from the fossils, including Picea burtonii , do not have united traces, cone axis to facilitate seed release (Smith and Stockey, 2001; some abietoid fossils do, including (Pityostrobus leckenbyi + Farjon 1998), which was likely accomplished in Picea burtonii P. oblongus ) and P. ramentosa Miller, as do several basal pinoids, via spreading of the ovuliferous scales. Nor does Picea burtonii namely Pityostrobus hallii Miller, P. villerotensis Alvin, and exhibit a dissected vascular cylinder, as seen in Abies , Cedrus , P. virginiana Robison et Miller (Alvin, 1957a; Creber, 1960; Pseudolarix, and several abietoid fossils including Obirastro- Miller, 1974 , 1976b ; Robison and Miller, 1977 ; Smith and bus Ohsawa, Nishida, et Nishida (1992), Pityostrobus milleri Stockey, 2001). Of the fossils that seem closely related to Pinus Falder, Rothwell, Mapes, Mapes, et Doguzhaeva (1998), and ( Gernandt et al., 2011 ; Fig. 4 ), only Pityostrobus lynnii has Pseudoaraucaria ( Miller, 1976a ; Smith and Stockey, 2001 , bract-scale traces that are united at their origin. Thus, it is prob- 2002 ). It should be noted, however, that dissected steles also able that extant Pinus merely refl ects an ancestral condition occur in Pityostrobus mcmurrayensis Stockey (1981) , P. pube- shared with extinct abietoids, and this character state should not scens Miller (1985) , and (P. beardii Smith et Stockey [2001] + be considered apomorphic for the Pinus lineage. P. hokodzensis Ratzel, Rothwell, Mapes, Mapes, et Doguzhaeva Miller (1976a ) demonstrated that the course and distribution [2001] ), all of which have been resolved as basal within of resin canals within the ovuliferous scale is diagnostic among Pinaceae (Gernandt et al., 2011; Fig. 4). While this may indi- extant Pinaceae and suggested that comparative assessment of cate that stele dissection is ancestral, it is possible that some the resin canals in fossil cones could offer phylogenetic insights. dissection is correlated to cone immaturity. During our reinves- In most nonabietoid cones, including Picea burtonii , two resin tigation of the Miller Conifer Reference Collection (Morpho- canals diverge from the vertical resin canal system on either side Bank P632), we found that when Picea cones appear to exhibit of the diverging bract-scale complex and accompany the vascu- dissected steles, the specimens are invariably immature. lar trace of the ovuliferous scale. Distal to the seed body, the Picea burtonii can be most confi dently excluded from the resin canals of the new cone also refl ect the character state com- abietoid lineage by its lack of two putative synapomorphies, the mon to most Pinaceae, in that resin canals are found both abax- first of which unites the terminal Pseudoaraucaria clade ial and adaxial to the vascular strands and between the vascular ( Alvin, 1957a , 1957b , 1960 , 1988 ; Miller and Robison, 1975 ). bundles. However, at the base of the ovuliferous scale, P. burtonii Although most pinaceous conifers exhibit an interseminal ridge, exhibits four resin canals adaxial to the vascular tissue, of which in Pseudoaraucaria it distinctly overarches the seed body. the upper two persist in an adaxial position, and ultimately be- While large interseminal ridges are found in some (i.e., come associated with the interseminal ridge. The lower two canals Gernandt et al., 2011), the two conditions appear independently become oriented abaxial to the vascular tissue and undergo 1074 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 2. Picea burtonii sp. nov. Holotype UAPC-ALTA P16098. (A) Radial longitudinal section of cone showing ovuliferous scale (os) and bract (b). Note inverted winged ovule borne on adaxial surface of ovuliferous scale. Bbot #15, scale = 500 µm. (B) Tangential longitudinal section through outer cortex of cone axis, showing proximal arrangement of vascular tissue and resin canals to the ovuliferous scales (os) and bracts (b) Bbot #9, scale = 1 mm. (C) Transverse section through bract-scale complex proximal to cone axis, showing abaxially concave vascular trace to ovuliferous scale (os), and terete bract trace (b). Bbot #26, scale = 50 µm. (D) Secondary xylem tracheids with helical to scalariform thickenings. Bbot #13, scale = 25 µm. (E) Transverse section through bract-scale complex at level of seed body. Note lateral separation of bract from ovuliferous scale and interseminal ridge. Ctop #2, scale = 500 µm. (F) Transverse section through bract-scale complex at chalazae. Ctop #40, scale = 500 µm. (G) Transverse section through bract-scale complex distal to seeds. Ctop #51, scale = 500 µm. June 2012] KLYMIUK AND STOCKEY—EARLIEST FOSSIL RECORD OF PICEA 1075 successive lateral divisions. This precise conformation is shared however, relaxed-clock estimates indicate early divergence only with extant Picea and with Pityostrobus oblongus (Creber, times for many of the extant genera ( Gernandt et al., 2008 ). 1960; Louvel, 1960; Smith and Stockey, 2001). Close affi nities Although cladistic analyses resolve Larix and Pseudotsuga between Picea burtonii and Pityostrobus oblongus may be con- as basal members of Pinaceae, they lack a Mesozoic fossil re- fi dently rejected, as this abietoid cone differs in a number of cord (Fig. 5). The oldest fossil assignable Larix is L. altoborea- characters, including markedly dilating cortical resin canals, lis LePage et Basinger emend. Jagel, LePage et Jiang, which bract and scale traces that are united at their origin, three separate has been described from fertile and vegetative remains from the origins of resin canals associated with the bract-scale complex, a Lutetian (middle Eocene) Buchanan Lake Fm of Axel Heiberg bract lacking an abaxial lobe, and resin vesicles within the seed Island ( LePage and Basinger, 1991 ; Jagels et al., 2001 ). While integument (Fliche, 1896; Creber, 1960; Louvel, 1960). Larix is well represented in Paleogene fl oras, the fossil record With respect to all anatomical and morphological characters of its sister taxon, Pseudotsuga, is limited to a single seed cone, assessed in our cladistic analyses, the new cone fully accords Pseudotsuga jechorekiae Czaja (2000) from the Miocene of only with the OTU Picea in the combination of characters it Germany. However, dispersed anatomically preserved leaves exhibits. Additionally, pollen grains associated with the cone that are similar to Pseudotsuga have been observed at Apple provide corroborative evidence for an affi nity with Picea , as Bay ( Stockey and Wiebe, 2006 ); thus Pseudotsuga may have a they can be defi nitively attributed to the genus (A. R. Sweet, broader stratigraphic range, congruent with recent phylogenetic Geological Survey of Canada, personal communication). Picea hypotheses ( Gernandt et al., 2011 ; Fig. 4 of this study). pollen grains are generally larger than those of other genera that Representatives of modern abietoid genera include Pseudola- possess bisaccate grains, with the exception of Abies ( Kapp, rix fossils from Cretaceous through Paleogene strata of Asia, 1969 ; Kapp and King, 2000 ). Abies pollen is distinguished from North America, and Europe ( LePage and Basinger, 1995 ), as that of pinoids by the triradiate streak upon its cappa (Bagnell, well as the fossilized woods, Cedrus penzhianaensis Blokhina 1975); like that of other pinoids, the cappa of the Apple Bay et Afonin (2007) and Keteleerioxylon kamtschatkiense Blokh ina, pollen is scabrose to psilate. Pollen found within the micropyles Afonin, et Popov (2006) from the Albian-late Albian (Lower Cre- of P. burtonii seeds is ~75 µm in total length, in accord with the taceous) Kedrovskaya Fm of the Russian Kamchatka Peninsula. lower end of the Picea size range ( Kapp, 1969 ). As in extant Another fossil wood, Abietoxylon shakhtnaense Blokhina Picea , the corpus of the P. burtonii pollen is ellipsoidal and (2010), from the upper Oligocene-lower Miocene of Sakhalin, suboblate, and sacci are borne low with respect to the corpus constitutes the earliest representative of an Abies- like plant. equator. This is typical of pinoid pollen, whereas in Abies , the However, these vegetative remains could also be attributed to sacci are borne nearly lateral to the corpus (Bagnell, 1975). The Pityostrobus taxa with abietoid affi nities. Like Abies , the fossil juncture formed between the corpus and sacci of the P. burtonii record of Tsuga is also relatively recent, in that isolated seeds, pollen forms an obtuse angle, which is characteristic of both scales and vegetative remains attributed to Tsuga are not repre- Picea and Cathaya ( Kapp and King, 2000 ; Liu and Basinger, sented prior to the Eocene ( LePage, 2003b ), although a Tsuga - 2000 ; Saito et al., 2000 ), whereas in Pinus , the angle is acute type palynomorph is known from the Upper Cretaceous of (Kapp, 1969; Kapp and King, 2000). Finally, the pollen exhib- Poland ( Macko, 1963 ). its sculptured cappula ridges that largely occlude the germinal Pinus belgica Alvin (1960), a seed cone presumed to be from furrow on the ventral, or distal, surface of the grain; occlusion the mid-Barremian or early Aptian Wealden Fm of Belgium, of the germinal furrow is diagnostic for Picea ( Bagnell, 1975 ). has been considered the oldest representative of both Pinus , and Preservation of pollen grains within ovules of pinaceous subfamily Pinoideae (Miller, 1976a ). In spite of uncertainty cones is rare, having previously been reported only in Pityostro- regarding the provenance of P. belgica (Gernandt et al., 2008; bus yixianensis Shang, Cui et Li (2001) , described from the P. Ryberg, University of Kansas, personal communication), the Aptian Jiufotang Formation of northeastern ( Shang et al., antiquity of the Pinus lineage is corroborated by several related 2001 ; He et al., 2004 ; Jiang and Sha, 2006 ). Although the pol- Aptian Pityostrobus species (Creber, 1956, 1967 ; Falder et al., len preserved in P. yixianensis is said to be similar to Picea , 1998). The stratigraphic range of Pinus itself can be extended to the cone itself differs from both extant Picea and P. burtonii 131–132 Ma, as an ovulate cone is known from the Speeton with respect to the course and distribution of resin canals and Clay Fm of Yorkshire ( Ryberg et al., 2010 ; P. Ryberg, personal vascular strands within the ovuliferous scale and lacks a keeled communication). bract (Shang et al., 2001). Furthermore, diagnostic features of In comparison to Pinus, the fossil records of other pinoid Picea pollen grains, like occlusion of the germinal furrow genera are surprisingly recent (Fig. 5). The earliest record for (Bagnell, 1975), are not apparent in specimens fi gured by Cathaya is a palynomorph, C. gaussenii Sivak, from the Lute- Shang et al. (2001). tian Buchanan Lake Fm ( Liu and Basinger, 2000 ). Like Ca- thaya, the earliest fossil record for Picea is also a palynomorph, Extant genera in paleontological context— Although P. grandivescipites Wodehouse, described from the Danian- Pinaceae is known to have evolved by the late Jurassic ( Miller, Selandian Fort Union Fm (Paleocene) of Montana’s Powder 1988 ; Rothwell et al., 2012 ), unequivocal representatives of River Basin (Wilson and Webster, 1946). To date, the oldest extant genera have not previously been reported from strata older Picea seed cone macrofossils are P. sverdrupii LePage, P. nansenii than the Barremian stage (~123 Ma) of the Early Cretaceous LePage, and P. palustris LePage, described from the Buchanan (Alvin, 1960; Fig. 5). While some authors consider detached Lake Fm (LePage, 2001). However, a Picea -like wood, Pice oxy- ovulate cone scales, seeds, and vegetative remains from Mon- lon talovskiense Blokhina et Afonin (2009) is known from golia’s Gurven-Eren Formation representative of the extant abi- the Albian of Russia, and Picea -like leaves, Midoriphyllum etoid genus Pseudolarix ( Krassilov, 1982 ; LePage and Basinger, piceoides, have been described from Apple Bay (Stockey 1995 ; LePage, 2003a ), both the pinaceous nature of this late and Wiebe, 2008). Thus, there is a stratigraphic disparity be- Jurassic taxon and its assignment to an extant genus is suspect tween fossils of Picea -like vegetation and the oldest cones (Rothwell et al., 2012). In contrast to the known fossil record, previously attributed to the genus, indicating that Picea cones 1076 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 3. Picea burtonii sp. nov. Holotype UAPC-ALTA P16098. (A) Longitudinal section through ovuliferous scale showing inverted winged ovule, with micropyle (m) oriented toward cone axis. Note presence of megagametophyte (mg), nucellar (n) tissue, and bisaccate pollen (p). Bbot #13, scale = 500 µm. (B) Longitudinal section through micropyle of ovule, containing several bisaccate pollen grains (p). Bbot #12, scale = 50 µm. (C) Longitudinal section through chalazal end of ovule, showing attachment of nucellus (n) to endotesta, and bisaccate pollen (p) embedded in apex of nucellus. Bbot #12, scale = 50 µm. (D) Bisaccate pollen grains in lateral and dorsal view. Sacci are borne at obtuse angle (arrow) to corpus, and exhibit prominent endoreticulations, which become fi ner at juncture of sacci with corpus. Bxs #72, scale = 25 µm. (E) Bisaccate pollen grains. Sacci associated with corpus (in ventral view, at arrow) broken away, but note germinal furrow (at arrow). Bxs #72, scale = 25 µm. are under-represented in paleobotanical collections. The new minimum divergence estimates were found for all nodes than ovulate cone from Apple Bay extends the stratigraphic range when the calibration was set at the Pseudolarix - Tsuga node, for Picea cones by ~75 Ma, bringing the fossil record for using Pseudolarix erensis Krassilov (1982) . Because the taxo- reproductive and vegetative organs with affi nities to Picea nomic affi nities of these latter fossils have been questioned into better accord. ( Rothwell et al., 2012 ), this calibration point may have inaccu- rately infl ated estimates of the family’s age. Implications of Picea burtonii for divergence estimates — Accurately estimating divergence within Pinaceae is a con- Fossil-calibrated relaxed-clock molecular divergence analyses siderable challenge due to the paucity of reliably dated fossils have yielded minimum divergence estimates for Pinaceae at that are readily assignable to extant genera (Gernandt et al., either 184 or 136 Ma (Gernandt et al., 2008), and long branches 2008). While recent assessments of fossil seed cones have dem- are evident at all basal nodes, particularly those subtending the onstrated that some Pityostrobus taxa may represent early pinoids ( Gernandt et al., 2008 ). When the fi xed-point fossil members of the abietoid and Pinus lineages, their use in calibra- calibration was set at 123 Ma for the Pinus-Picea node, using tion is confounded by stratigraphic imprecision (Fig. 5). In the early Aptian seed cone, Pityostrobus bommeri , younger some cases, the collecting localities are unknown (i.e., Alvin, June 2012] KLYMIUK AND STOCKEY—EARLIEST FOSSIL RECORD OF PICEA 1077

Fig. 4. Nelson consensus cladogram of two equally parsimonious trees (L = 262, CI = 0.24, RI = 0.59) produced from analysis of 54 morphological characters (after Gernandt et al., 2011 ) using the parsimony ratchet perturbation algorithm ( Nixon, 1999 ), with bootstrap support values given at nodes. Fossil taxa are indicated by (†).

1960), and in others, the stratigraphic provenance is poorly con- oldest anatomically preserved pinaceous seed cones ( Fig. 5 ), its strained (i.e., Ohsawa et al., 1992 ). Finally, many of these cones attribution to an extant genus not only resolves a ghost lineage have been described from sedimentary successions that are not indicated by sequence divergence estimates ( Gernandt et al., reliably dated. A less conspicuous problem infl uencing accu- 2008), but provides important new data for calibrating these racy is the use of fossils as point calibrations (e.g., Gernandt hypotheses. et al., 2008), which implies a direct ancestor-descendent rela- tionship between the taxon and a crown group ( Ho and Phillips, Evolution of Picea — Today, Picea comprises ~38 species, 2009 ), and can produce dramatically different divergence time only eight of which occur in North America, with the remainder estimates (Doyle and Donoghue, 1993; Magallón and Sanderson, restricted to Asia ( Farjon, 2010 ). Phylogenetic analyses of 2001 ; Forest et al., 2005 ). cpDNA, including matK , trnT-L-F ( Germano et al., 2002 ), and Effects of stratigraphic errors and/or incorrect placement of a trnC-trnD (Ran et al., 2006), in addition to the mitochondrial calibration taxon can be reduced by using multiple calibration nad5 intron ( Ran et al., 2006 ), have resolved two North Ameri- points ( Soltis et al., 2002 ; Conroy and Van Tuinen, 2003 ; Graur can species, Picea sitchensis (Bongard) Carrière and P. brewe- and Martin, 2004; Near et al., 2005), and future work at Apple riana S. Watson, as the most basal members of the extant genus. Bay may reveal other fossils attributable to extant lineages. Although introgression is common among living spruce, Several leaf morphologies have been identifi ed at the locality, cpDNA haplotypes in Picea are typically species-specifi c ( Du including Tsuga -, Pseudotsuga- , Pinus-, and Abies- like forms et al., 2009 ). Therefore, these fi ndings indicate a North (Stockey and Wiebe, 2006), in addition to the Picea -like American origin for the genus (Sigurgeirsson and Szmidt, Midoriphyllum piceoides (Stockey and Wiebe, 2008). It is 1993 ; Ran et al., 2006 ), which is corroborated by the pres- therefore apparent that similar to extant genera had ence of a number of Palaeogene fossils attributable to Picea evolved and diversifi ed by the Valanginian and that pinaceous (reviewed by LePage, 2001 ). Picea burtonii , as the earliest diversity in the Early Cretaceous has been both undersampled known member of the spruce lineage, provides further support and underestimated. As Picea burtonii represents one of the for this biogeographic hypothesis. 1078 AMERICAN JOURNAL OF BOTANY [Vol. 99 June 2012] KLYMIUK AND STOCKEY—EARLIEST FOSSIL RECORD OF PICEA 1079

Although Picea burtonii exhibits combinations of characters some species of Picea exhibit the trait ( Stockey and Wiebe, that can be found within Picea , it does not fully accord with any 2008 ), as does Cathaya ( Hu and Wang, 1984 ), which cladistic extant species (Morphobank P632). It is also distinct from the analyses suggest is sister to Picea (Gernandt et al., 2008, 2011 ; three other anatomically preserved Picea seed cone fossils, all Fig. 4). Transfusion tissue in M. piceoides is like that shared by of which have been described from the Cenozoic of North Picea and Pinus, in that it completely surrounds the vascular America ( Miller 1970 , 1989 ; Crabtree, 1983 ). Two Oligocene bundle ( Hu and Yao, 1983 ; Stockey and Wiebe, 2008 ) and is species, P. diettertiana Miller (1970) and P. eichornii Miller therefore derived with respect to that of Cathaya, in which it (1989) , were described from western Montana and the Olympic occurs only lateral and abaxial to the vascular bundle ( Hu and Peninsula of Washington State, respectively. A Serravalian Wang, 1984 ), as in extant abietoids and the basal sister-groups (Miocene) species, P. wolfei Crabtree (1983) , has been described Larix and Pseudotsuga (Hu and Yao, 1983; Hu and Wang, from northwestern Nevada. 1984). If Midoriphyllum piceoides and Picea burtonii are veg- Picea burtonii differs from both P. diettertiana and etative and reproductive organs, respectively, of the same plant, P. eichornii , in that the pith of the P. burtonii cone axis contains we can infer that evolution within Picea occurred in a mosaic sclereids, the inner cortex lacks sclerenchyma, and there are no fashion, whereby anatomically modern seed cones were borne resin canals associated with the secondary wood of the vascular by plants with more primitive vegetation. cylinder. Picea burtonii is most obviously differentiated from However, the reconciliation of these two taxa as an organis- P. diettertiana by the conformation of the resin canal system mal concept for a Valanginian spruce may ultimately be precluded associated with the ovuliferous scale. When the scale of P. diet- by the nature of the depositional setting within which M. pi- tertiana is examined at the level of the seed, only two resin ceoides and P. burtonii are preserved. Plant fossils at Apple canals occur abaxial to the vascular tissue, whereas in most spe- Bay are preserved as allochthonous phytodetrital components cies of Picea , including P. burtonii , the abaxial resin canals lat- of probable tempestites. Such marine assemblages are often erally ramify. Similarly, in most Picea cones, the resin canals products of time averaging (Slatt, 1974; Flessa et al., 1993; persist beyond the level of the seed (Morphobank P632), Kidwell, 2002; Kowalewski, 1996), and it remains to be deter- whereas in P. diettertiana , they do not ( Miller, 1970 ). Both mined whether paraconformities between individual beds re- Picea diettertiana and P. wolfei have predominantly parenchy- fl ect periods of non-deposition or deposition and subsequent matous bracts, with sclerenchyma restricted to a narrow zone reactivation. adaxial to the vascular tissue, whereas in P. eichornii , the bract is described as having sclerenchyma identical to, and continu- Further research — The late Mesozoic was a time of rapid ous with, that of the outer cortex ( Miller, 1989 ). The bracts of evolution and radiation of Pinaceae, and although the Pityostro- P. burtonii are predominantly parenchymatous, and there is no bus assemblage represents a period of great diversity within the evidence of sclerenchyma adaxial to the bract trace, as in family, these extinct conifers demonstrably coexisted with early P. diettertiana and P. wolfei . members of extant lineages. As such, our understanding of the Of the Cenozoic fossils, Picea burtonii is most similar to the family’s phylogeny is dependent upon clarifi cation of the evo- Miocene cone, P. wolfei in terms of sclerenchyma and resin lutionary polarity of anatomical characters used to assess fossil canal distribution within the cone axis ( Crabtree, 1983 ). How- seed cones. Increased recognition of the degree of conservation ever, the broad, fl attened ovuliferous scale trace of P. wolfei is in gene families controlling development and expression of more robust than that of P. burtonii , wherein the vascular trace seed cone characters, and a better understanding of the extent to rapidly divides into a number of delicate strands. Additionally, which cone morphology is infl uenced by epigenetic effects, the two resin canals associated with the interseminal ridge of may ultimately provide a theoretical framework for interpreting P. burtonii persist unaltered throughout the scale, whereas in anatomical characters, which can then be interpolated to the P. wolfei they ramify laterally distal to the seed body, produc- fossil record. Such advances may permit appropriate weighting ing an adaxial series of 10–12 smaller canals ( Crabtree, 1983 ). hypotheses, allowing accurate representation of phylogenetic The similarities between Picea burtonii and Cenozoic and signals embodied by the morphological and anatomical charac- extant species suggest that seed cone morphology has been ters used to assess pinaceous fossils. highly conserved over the ~136 Ma history of the genus. In It should be noted, however, that comparative anatomy for comparison, the Picea -like leaf described from Apple Bay living plants has been only sporadically investigated (Crane (Stockey and Wiebe, 2008) exhibits a combination of charac- et al., 2004). As the most complete compilation of anatomical ters that may refl ect an ancestral condition. The vascular bundle sections, the Miller Conifer Reference Collection encompasses and resin canal anatomy of Midoriphyllum piceoides , as well as only a fraction of the extant species of Pinaceae and fewer than the thickened hypodermis associated with leaf angles, are very half the living species of Picea . While continued investigations similar to extant Picea (Stockey and Wiebe, 2008). The pro- of Early Cretaceous and Late Jurassic deposits are likely to expand nounced plicate mesophyll is more typical of Pinus , although our understanding of the evolutionary history of Pinaceae, it is

← Fig. 5. Diversity of pinaceous fossils, correlated to stratigraphic provenance. Stratigraphy is adapted from the Internationa l Commission on Stratigra- phy (2010), with y -axis proportional to time (Ma), at right. In addition to seed cone macrofossils, the earliest known exemplars of extant genera are repre- sented, as are Picea fossils that exhibit anatomical preservation. “Pseudolarix erensis” is not thought to be a valid taxonomic designation. Error bars refl ect uncertainty of provenance and imprecision in dating sedimentary strata from which the fossils have been described ( Coemans, 1866 ; Fliche, 1896 ; Dutt, 1916 ; Seward, 1919 ; Wilson and Webster, 1946 ; Alvin, 1953 , 1957a , 1957b , 1960 , 1988 ; Creber, 1956 , 1960 , 1967 ; Louvel, 1960 ; Macko, 1963 ; Miller, 1970 , 1972 , 1974 , 1976b , 1977b , 1978 , 1985 , 1989 ; Miller and Robison, 1975 ; Robison and Miller, 1977 ; Stockey, 1981 ; Krassilov, 1982 ; Crabtree, 1983 ; Crabtree and Miller, 1989 ; LePage and Basinger, 1991 ; Ohsawa et al., 1991 , 1992 ; Falder et al., 1998 ; Czaja, 2000 ; Liu and Basinger, 2000 ; LePage, 2001 ; Ratzel et al., 2001 ; Shang et al., 2001 ; Smith and Stockey, 2001 , 2002 ; Blokhina et al., 2006 ; Blokhina and Afonin, 2007 ; Blokhina, 2010 ; Ryberg et al., 2010 ). 1080 AMERICAN JOURNAL OF BOTANY [Vol. 99 imperative that the range of variation within extant genera be C ZAJA , A. 2000 . 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