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Anatomy and Morphology |
2Department of Botany, The Field Museum of Natural History, Chicago, Illinois 60605-2496 USA; 3School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501 USA; 4Laboratory of Systematic and Evolutionary Botany and Herbarium, Institute of Botany, Chinese Academy of Sciences, Beijing 100093 People's Republic of China
Received for publication November 10, 2004. Accepted for publication May 16, 2005.
ABSTRACT
The sweet gum genus Liquidambar (Altingiaceae) has two species in eastern Asia, one in eastern North America, and one in western Asia. Mature infructescences are studied to provide anatomical, morphological, and micromorphological details, some of which are newly recognized. Homology is suggested between extrafloral spinose processes of L. formosana and L. acalycina, braid-like ornamentation of L. styraciflua, and broad intercarpellate areas of L. orientalis. Morphology, position, number, and the presence of similar structures in the closely related Hamamelidaceae s.s. support their derivation from sterile flowers. Morphological cladistic analysis using 43 characters supports the monophyly of Liquidambar with Altingia as its sister. The matK analysis contrastingly places Altingia sister to the L. acalycina–L. formosana clade, rendering Liquidambar paraphyletic. Discordance between morphological and matK data sets may result from both different rates of morphological evolution and convergence. Several similarities between Altingia and L. acalycina are symplesiomorphic in the morphological cladistic analysis. Microaltingia apocarpela, from the Cretaceous of eastern North America, documents the earliest known fossil divergence within Altingiaceae. The Miocene Liquidambar changii of western North America is sister to a clade of extant Liquidambar species. Consideration of this fossil evidence reveals complex intercontinental biogeographic disjunctions in Altingiaceae.
Key Words: Altingiaceae • biogeography • infructescence • Liquidambar • phylogeny
The family Altingiaceae Horan., with its approximately 15 tree species and excellent fossil record, is an important model for understanding northern hemisphere biogeography. The most commonly known genus, Liquidambar L. (Altingiaceae), contains four intercontinentally disjunct species in the temperate zone of the northern hemisphere. Two species are in eastern Asia, one is in western Asia, and one is disjunct between eastern North America and from central Mexico to Belize (Fig. 1). These arborescent taxa have unisexual, capitate, globose, woody infructescences composed of 25–50 helically arranged, bilocular capsules that bear several viable seeds per fruit and many abortive seeds. Liquidambar has been the focus of several studies exploring the evolution of intercontinental biogeographic disjunctions in the north temperate zone (see review in Wen, 1999
). Allozyme (Hoey and Parks, 1991
, 1994
) and phylogenetic analyses of molecular sequencing data (Shi et al., 1998
; Li et al., 1997a
, b
; Li and Donoghue, 1999
) suggest a sister species relationship between the North American L. styraciflua L. and the western Asian L. orientalis Mill. within the genus. This hypothesized relationship suggests an historical connection across the North Atlantic, rather than a Eurasian route. Liquidambar orientalis may have diverged from relatives of L. styraciflua of North America prior to the disruption of the North Atlantic land bridge, around 45 million years ago (mya) (Graham, 1999
; Tiffney and Manchester, 2001
).
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; Reinsch, 1890
; Chang, 1979
; Cronquist, 1981
; Endress, 1989a
; Qui et al., 1998
; Li and Donoghue, 1999
) or (incorrectly) as Liquidambaroideae (Harms, 1930
; Bogle, 1986
; Ferguson, 1989
). Some authors have elevated the subfamily to family status as the Altingiaceae (Blume, 1828
; Wilson, 1905
; Chang, 1959
; Rao, 1974
), a taxonomic level accepted today by many (e.g., Judd et al., 2002
; APG II, 2003
).
The systematic composition of the Altingiaceae has been relatively stable, while its systematic position has been in flux. Traditionally, Hamamelidaceae s.l. (including Altingiaceae) has been considered a member of the Hamamelididae Takht. (Cronquist, 1981
). Recent molecular studies have shown this assemblage to be polyphyletic and support Altingiaceae and Hamamelidaceae s.s. as members of the saxifragoid clade within a larger rosid clade (Chase et al., 1993
; Magallón et al., 1999
; Soltis et al., 2000
; APG II, 2003
). In an alternative classification, the order Altingiales, including Altingiaceae and Rhodoleiaceae, was recognized and included in the superorder Trochodendranae along with the order Trochodendrales (Doweld, 1998
). Recognition of the four species of Liquidambar has been widely accepted; however, a few authors have suggested that L. styraciflua and L. orientalis might be conspecific based on the continuous, clinal variation among leaves and on the relatively subtle morphological differences between their infructescences (Reichinger, 1943
; Meikle, 1977
; Ferguson, 1989
). The similarity between these two species may also be due to morphological stasis that is thought to occur among many temperate intercontinental disjunct genera (Wen, 1999
). Stasis is supported by the morphological similarities in the two species, despite their high level of molecular divergence (Wen, 1998
, 1999
, 2001
). Morphological stasis has also been proposed for other disjunct genera including Liriodendron L. (Parks and Wendel, 1990
), and Magnolia L. sect. Rytidospermum (Qui et al., 1995
). We now know that a fossil species of Liquidambar sharing many similarities with the extant Asian species L. acalycina was present by the middle Miocene (15 mya) (Pigg et al., 2004
), suggesting that an earlier migration of ancestors of L. styraciflua and L. orientalis is realistic.
In the present study we describe comprehensive details of infructescence morphology, micromorphology, and anatomical structure of the four extant species of Liquidambar. This study was undertaken to better understand the overall morphological diversity, character evolution, and delimitation of extant species within the genus, and the biogeographic implications of these characters. Detailed comparable analyses of the other two genera of Altingiaceae, Altingia and Semiliquidambar, and of related fossil taxa, are currently underway within a broader study to detail the phylogeny, morphological diversification, evolutionary history, and biogeography of the Altingiaceae.
MATERIALS AND METHODS
Taxa for study were obtained by fresh or herbarium sample and photographed for general features (Appendix). Measurements given are the mean of 10 individuals counted (Table 1). Specimens for anatomical sections were prepared using standard histological techniques that included embedding in Paraplast Plus tissue embedding medium (Monoject Scientific, St. Louis, Missouri, USA) and sectioning on a rotary microtome at 20 µm thick. Mature infructescences were softened with ethylene diamine prior to embedding (Carlquist, 1982
). For anatomical studies, dry seeds were rehydrated for 7 d in equal parts of glycerol, water, and ethanol and then sectioned by hand (Lobova et al., 2003
). Seeds and carpel wall surfaces were prepared for scanning electron microscopy by sputter coating and scanned with an Amray 1400 and Amray 1810 SEM (KLA Tencor, Bedford, Massachusetts, USA). Except where noted, terminology follows that of Bogle (1986)
.
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), and the middle Miocene, Liquidambar changii Pigg, Ickert-Bond & Wen from western North America (Pigg et al., 2004
). The Altingiaceae have occupied an isolated position in the saxifragoid clade, which makes outgroup selection difficult. Exbucklandia R. W. Brown and Hamamelis L. of Hamamelidaceae s.s. (also in the saxifragoid clade) were selected as the outgroups in our analysis following earlier suggestions (Table 3). Semiliquidambar was not included in the analysis because it has been hypothesized to be an intergeneric hybrid (Bogle, 1986
; Ferguson, 1989
). We emphasized reproductive structures and expanded the established morphological matrix of Hufford (1992)
, and also included the characters of chromosome number and iridoid chemistry. Forty-two characters were selected on the basis of interspecific variations among the sampled taxa (Table 3). They consist of 38 binary and four multistate characters. All multistate characters were treated as unordered. Quantitative characters were coded following simple gap coding (Archie, 1985
). Characters for which discrete states could not be recognized (i.e., continuous characters) were excluded from the data matrix. Data are mainly derived from our own observations and partly from the literature (Chang, 1962
; Melikian, 1973
; Kaul and Kapil, 1974
; Goldblatt and Endress, 1977
; Bogle, 1986
; Endress, 1989a
, b
, c
; Ferguson, 1989
; Hufford and Endress, 1989
; Hufford and Crane, 1989
; Endress and Stumpf, 1990
; Hufford, 1992
). An annotated list of all characters is presented in Table 2 and the morphological matrix is presented in Table 3.
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). Character states were coded as unordered and all characters were weighted equally. The amount of support for monophyletic groups revealed in the maximally parsimonious tree(s) (MPTs) was examined with 1000 bootstrap replicates (Felsenstein, 1985
) with the random addition and the heuristic search options.
We used matK sequences generated from a recently published phylogenetic study of Altingiaceae (Shi et al., 2001
) as a molecular framework for discussing our morphological evolution in Liquidambar and close relatives. Our preliminary results from analysis of waxy and trnL-trnF generated the same topology for these taxa as in the matK tree (Ickert-Bond et al., 2004
). For the matK data set, we obtained the sequence data from GenBank: Liquidambar styraciflua (AF133219), L. acalycina Hung T. Chang (AF133222), L. formosana Hance (AF015650), L. orientalis (AF133220), A. chinensis (AF133225), Exbucklandia populnea (AF128831), and Hamamelis virginiana (AF013046). We executed the phylogenetic analysis with both parsimony and Bayesian methods. Base frequencies were empirically determined, and the transition: transversion (K) ratio and gamma shape parameter (
) were estimated on MP trees under the model of sequence evolution, chosen using results from ModelTest version 3.06 (Posada and Crandall, 1998
). Bayesian inference was conducted using MrBayes version 3.0 (Huelsenbeck and Ronquist, 2001
). The Markov chain Monte Carlo algorithm (MCMC) was run for 2 000 000 generations with four incrementally heated chains, starting from random trees and sampling one out of every 100 generations. A majority-rule consensus tree was calculated with PAUP* from the last 18 001 out of the 20 001 trees sampled. The first 2000 trees (burn-in) were excluded to avoid trees that might have been sampled prior to convergence of the Markov chains. The posterior probability of each topological bipartition was estimated by the frequency of these bipartitions across all 18 001 trees sampled. Internodes with posterior probabilities 
95% were considered statistically significant.
The relationships of the morphological analysis were tested against alternative hypotheses. Trees with topological constraints were constructed using MacClade 4.03 (Maddison and Maddison, 2000
), then loaded into PAUP*. Heuristic searches were conducted to find the shortest trees consistent with each imposed constraint. The following three hypotheses were tested: (1) monophyly of Liquidambar acalycina and L. formosana; (2) matK phylogeny; and (3) monophyly of the eastern Asian Altingia chinensis, L. acalycina, and L. formosana. The additional steps required for a particular constraint are given by the difference in length between the shortest trees obtained for the constrained versus unconstrained tree.
We employed the partition homogeneity test (Farris et al., 1995
) and the Templeton's test (Templeton, 1983
) to evaluate the congruence of the morphological and molecular data sets. The partition homogeneity test was conducted with PAUP* with 100 replicates of heuristic search using TBR branch-swapping and gaps treated as missing data. Topological congruence between the gene trees was evaluated with the Templeton's test, as implemented in PAUP*.
RESULTS
Morphological description
General features
The pistillate reproductive structures in Liquidambar are characterized by a spherical inflorescence (Figs. 2C, 4B, 5B, 6B) that develops into a persistent, woody infructescence (Figs. 2A, 3A, 4A, 5A, E, 6A). It is composed of closely spaced, multiple (
25–50) bilocular fruits that appear to be helically arranged around a central axis borne on an elongate peduncle. Extrafloral structures that have been variably interpreted are found within the infructescence head. Depending on the species under study they include, variously, elongate, spine-like processes (L. acalycina, L. formosana, Figs. 3B, 4C), thickenings that appear organized into a tangentially elongate "braided" structure along the hypanthial surface (L. styraciflua, Figs. 2A, 6D), or smooth, slightly thickened areas between adjacent fruits (the "peripheral rim" sensu Gregor, 1978
of L. orientalis; Ferguson, 1989
; Fig. 5E).
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The bilocular fruits are wedge- to spindle-shaped, fused basally, and free distally (Figs. 2B, 4C, 6I). In some taxa, individual fruits are tightly connected to one another, resulting in a cohesive infructescence (Figs. 4E, 5D, 6H); in others, they are only loosely attached to one another, and the infructescence falls apart into individual fruits upon hand-sectioning (Fig. 3C). Carpels are fused basally along their ventral margins. In this region the tissues typically become thickened and sclerified (Figs. 2E, 3D, F, 4F).
Fruits typically have a two-zoned pericarp consisting of a persistent inner carpel wall of palisade-like cells one to several cells thick (Figs. 2E, H, 3H, 4H, 5M, 6G) and an outer pericarp 20–30 cells thick (Fig. 2E, F). The inner palisade cells sometimes have thickened walls (Fig. 6G). To the outside of the palisade layer, the outer pericarp is made of approximately four layers of ground tissue and a uniseriate epidermis. The outer pericarp's innermost layer is a zone of 5–6 cells thick of tangentially elongate parenchyma cells (Figs. 2F, G, 3H, J). The second layer is an area of larger parenchymatous cells, 2–4 cells thick, which contains resin canals (Figs. 2F, 3J, 5M). This layer is often disrupted and often breaks down in mature fruits (Figs. 2G, 3F, 4F). Next are the vascular strands comprised of 2–5 primary xylem elements and 2–6 layers of phloem, surrounded by often fibrous bundle sheaths (Figs. 2 E, I, 5L). The outermost ground tissue is composed of 8–10 layers of parenchymatous cells, within which resin canals and crystals are found (Fig. 4L). A uniseriate epidermis is composed of tabular to radially elongate to papillate cells (Figs. 2E, 4K).
Infructescences are borne on a woody peduncle of varying length. The axis ranges from 0.9–2.0 mm in diameter with a central stele (1.1 mm in diameter) surrounding a broad pith and contains a small amount of secondary tissue surrounded by ground tissue that contains prominent resin canals (Fig. 2H). The stele extends into the central part of the infructescence where it appears as individual sympodia, each subtended by several rows of secondary tissues (Fig. 2H).
A large number of anatropous ovules are initially borne broadly in a vertical row along the ventral margins of each carpel (Figs. 2C, E, 6I). Typically, only a few ovules develop into mature seeds, and they tend to be found basally, closest to the central axis. Maturing seeds are either broadly obovate with a circular flange (Fig. 3K, L) or elongate to spindle-shaped with a prominent distal wing (Figs. 4M, 5C, 6F, 8A– D). They are typically speckled or striped and there is a wide variation in this feature, even within a single infructescence. The seed coat is several layers of cells thick, with a prominent outer layer of palisade cells and around 3–5 inner layers surrounding a straight embryo cavity. Numerous abortive seeds are variable in shape and have a prominent palisade layer that resembles the outermost seed coat of mature seeds, but otherwise they fail to develop.
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= 27.0) mm high by 18.4–31.1 ( = 26.9) mm wide, with a length/width (L/W) ratio ranging from 0.8–1.2: 1 (= 1.0 : 1). Peduncles are 53.2– 65.9 ( = 59.8 mm) long by 0.9–1.3 ( = 1.1) mm wide. Each infructescence is made of 26 individual bilocular fruits that appear to be helically arranged on a central axis.
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4.0–6.5 ( = 5.5) mm long and curving. The paired styles from each fruit curve toward one another at their tips (Fig. 3A, B). They gradually extend basally into elongate, triangular-shaped bases that expand to 3.4 mm wide. Surrounding each fruit are typically 10–12 short, spinose extrafloral structures, each up to 2.7 mm long x 0.3 mm wide that are attached to a wider cushion-like base 0.9 mm across (Fig. 3B). Both the styles and extrafloral structures are finely striated vertically.
Fruits are 8.0 mm long x 2.8 mm wide (L/W ratio of 2.9 : 1), wedge-shaped and fused for 2/3–3/4 of their total length, and free distally (Fig. 3C, D). Fruits are held together somewhat loosely (Fig. 3C), with a relatively thin pericarp, and spaces appear between adjacent fruits (Fig. 3E, F). In mature infructescences the pericarp is composed of an inner palisade layer two cells thick lining the locule (Fig. 3H), and a multicellular outer fruit wall (Fig. 3J). The palisade layer has two rows of radially elongate, thin-walled cells with individual cells ranging in size from 8–9 µm long x 2–11 µm wide (Fig. 3H). The outer fruit tissue is typically torn in mature infructescences, with some of the innermost layers adhering to the inner palisade layer (Fig. 3F). To the outside of the carpel lining, the next layer is a single layer of tabular cells that are slightly tangentially elongate and 8 µm long x 2 µm wide (Fig. 3F, H). To the outside is a zone of tangentially elongate cells around 6–8 cells thick (Fig. 3H). A zone 12–20 cells thick of more isodiametric cells makes up the central fruit tissue, with the outermost tissues containing vascular bundles subtended by prominent resin canals (Fig. 3J) and fiber bundles 5 µm wide that subtend the vascular bundles (Fig. 3J). The surface of the inner carpel wall has tangentially elongate cells, that appear rod-like and anastomosing (Fig. 7G).
Seeds are 5.8 mm long x 4.7 mm wide (L : W ratio, 1.8 :1) with a circular flange surrounding the central body, resulting in a somewhat triangular appearance (Figs. 3K, L, 9A). Cells of the seed coat are polygonal (4–5 sided) and slightly elongate, and appear slightly thickened on the anticlinal walls (Fig. 8E). The cuticle of the epidermis is thin and epidermal cells are tabular in cross-section (Fig. 9J). Below the epidermis is a layer of fibers with large lumina (Fig. 9E), followed by crushed epidermal cells of the inner integument. To the inside of this layer are parenchymatous cells of the endosperm (Fig. 9E). The embryo is straight (Fig. 9A).
The most distinctive features of this species are the paired, curved styles and spinose extrafloral structures (Fig. 3B), the loosely attached fruits (Fig. 3C), the prominent basal pad of tissue in the fruits (Fig. 3D), and the triangular, flanged seeds (Fig. 3K, L) with a polygonal seed coat pattern (Fig. 8E) composed of epidermal cells that are tabular in cross-section (Fig. 9J).
Liquidambar formosana (Figs. 2F, 4A–M, 7B, H, 8B, F, 9B, F)
Infructescences are spherical with persistent styles (Fig. 4A) and measure 24.3–31.0 ( = 27.2) mm high by 22.5–30.4 ( = 27.0) mm wide, with a L/W ratio ranging from 0.9–1.1 : 1 ( = 1.0 : 1). Peduncles are 38.9–63.1 ( = 55.2) mm long x 1.2–1.5 ( = 1.4) mm wide. Each infructescence is made up of 34 individual bilocular fruits that appear to be helically arranged on a central axis.
Styles are 6.8–9.8 ( = 8.2) mm long. When found in inflorescences, they have tightly coiled tips that resemble fern croziers or "fiddleheads" (Fig. 4D); at maturity within infructescences they become somewhat dried and brittle and slightly uncoiled, but remain tightly coiled at the very tip (Fig. 4C). Styles extend basally into abruptly broad bases that expand to 2.7 mm wide (Fig. 4C, I). Surrounding each is a group of around a dozen spinose extrafloral structures up to 6.9 mm long x 0.3 mm wide that resemble the styles except for lacking the coiled tips and stigmatic areas (Fig. 4C). These extrafloral structures each expand basally into a triangular base 1.5 mm across. They are fused together basally into elongate cylinders up to 8.5 mm long that greatly resemble the paired ovaries of each fruit (Fig. 4C). These structures are further fused together to form a band that surrounds a viable fruit (Fig. 4C).
Fruits are 8.7 mm long x 2. 2 mm wide (L/W ratio, 4 : 1), cylindrical to spindle-shaped, fused for 3/4 of their total length and free distally (Fig. 4E). Fruits are held together tightly and have relatively thin fruit walls (Fig 4E). The fruit wall is composed of an inner, uniseriate palisade layer (Fig. 4F, G, H) and an outer pericarp (Fig. 4K, L). Individual cells of the inner palisade layer are radially elongate with thin walls that may have obliquely oriented rather than entirely perpendicular end walls (Fig. 4H). Palisade cells range in size from 26–28 µm long x 6–14 µm wide. To the outside, a remnant of the outer pericarp adheres to the palisade layer (Fig. 4H). The organization of the outer fruit wall is similar to the general structures described earlier for L. styraciflua (Fig. 2G) and L. acalycina. Distinctive features of the pericarp in this species include numerous druse crystals in the outermost pericarp and epidermal cells that are extremely elongate radially (Figs. 2F, 4K, L). Surface features of the inner carpel wall have short cells, that appear slightly raised above the general surface (Fig. 7G).
Seeds are 7.7 mm long x 1.9 mm wide (L/W ratio, 4.0 :1) and very tapered, flattened in transverse section (Fig. 4G), and have a prominent distal wing that may be up to 3.8 mm long (Figs. 4M, 8B). Embryos found within seeds (Fig. 4G) are straight. Cells of the seed coat are tangentially elongated and rectangular with relatively thickened anticlinal walls in surface view (Fig. 8F). Epidermal cells of the seed coat are rectangular in cross-section and are covered by a thin cuticle. To the inside of the epidermis is a hypodermal layer that contains druse crystals, which in turn is followed by the outer integument of mostly crushed parenchyma cells. To the inside of the hypodermis are 2–3 layers of macrosclereids with small lumina (Fig. 9F), followed by the inner integument and the multilayered parenchymatous endosperm (Fig. 9F).
Features that are of particular diagnostic value for this species include the prominent extrafloral structures, the highly coiled style tips, the presence of druses in the outer fruit wall, the elongate seeds with a prominent distal wing (Fig. 4M), and 2–3 layers of macrosclereids in the seed coat (Fig. 9F).
Liquidambar orientalis (Figs. 5A–M, 7C, I, 8C, G, 9C, G, I)
Infructescences are spherical with persistent styles (Fig. 5A, E), and a broad, relatively smooth surface between adjacent fruits (Fig. 5E) and measure 12.2–26.6 ( = 14.9) mm high by 14.5–27.0 ( = 17.4) mm wide, with a L/W ratio ranging from 0.8–1.1 : 1 ( = 0.9 : 1). Peduncles are 14.0– 47.5 ( = 29.0) mm long x 0.9–1.5 ( = 1.2) mm wide. Each infructescence is made up of 48 individual bilocular fruits that appear to be helically arranged on a central axis.
Styles are 2.1–3.5 ( = 3.0) mm long. When found in inflorescences, styles are thick and fleshy, with a simple beak-like stigmatic tip (Fig. 5G). In mature infructescences, styles are persistent, with each attached to an elongate, triangular base up to 3.1 mm wide. They are somewhat vertically striated and may frequently be broken at the tip (Fig. 5E).
Fruits are 6.0 mm long x 2.5 mm wide (L/W ratio 2.4 : 1), cylindrical to slightly wedge-shaped (Fig. 5C), and fused for 2/3 to 3/4 of their total length and free distally. Fruits are held together fairly tightly, with relatively thin fruit walls. The fruit wall is composed of an inner palisade layer (Fig. 5K, M) and an outer pericarp (Fig. 5L). The palisade layer is a single cell thick. Individual cells are cuboidal to slightly radially elongate with thickened walls and range from 10–11 µm long x 3–5 µm wide (Fig. 5J). Surface features of the inner carpel wall show short, rectangular cells, with oblique end walls (Fig. 7I).
Seeds are 4.7 mm long x 1.9 mm wide (L/W ratio 2.6 : 1), elongate, and have a relatively short distal wing (Fig. 5C). The seed body is often irregular in shape, sometimes with a deep elongate groove (Fig. 5C). Cells of the seed coat are tangentially elongate, rectangular with thickened anticlinal walls, and oblique end walls (Fig. 8G). The epidermal cells are cuboidal in cross-section and are covered by a thick cuticle (Fig. 9G). Below the epidermis is a single-layered hypodermis containing druse crystals (Fig. 9I), followed by 2–3 layers of macrosclereids each with a small lumen (Fig. 9G). To the inside is the epidermis of the outer integument, followed by parenchymatous cells of the endosperm.
Characteristic features of this species include the thick, relatively smooth areas between individual fruits, the cylindrical to spindle-shaped carpels, relatively short styles, the large number of flowers per inflorescence and the elongate seeds with deep grooves and relatively short distal wings, a mulicellular layer of macrosclereids in the seed coat (Fig. 9G), and thickened anticlinal walls of the inner carpel wall.
Liquidambar styraciflua (Figs. 6A–N, 7D–F, J, 8D, H, 9D, H)
Infructescences are spherical with persistent styles and small knob-like protuberances on surfaces (Fig. 6A, C–D) and measure19.0–34.6 ( = 30.3) mm high x 20.3–40.9 ( = 31.6) mm wide, with a L/W ratio ranging from 0.8–1.1 : 1 ( = 0.97 : 1). Peduncles are 36.7–55.9 ( = 42.8) mm long by 0.9–1.1 ( = 0.95) mm wide. Each infructescence is made up of 37 individual bilocular fruits that appear to be helically arranged on a central axis.
Styles are 4.1–6.3 ( = 5.2) mm long. In inflorescences, the styles are fleshy and extend into short, hooked tips (Fig. 6B, E). Mature infructescences have styles that have become brittle with elongately striate triangular bases that expand to 4.9 mm wide (Fig. 6J). Within the areas between adjacent fruits are numerous knob-like extrafloral structures that have a "braided" appearance (Fig. 6C, D).
Fruits are 12.4 mm long x 4.3 mm wide (L/W ratio, 2.9 :1), cylindrical to spindle-shaped, fused for 2/3–3/4 of their total length, and free distally (Fig. 6H–I). Fruits are held together tightly (Fig. 6H), with relatively thin fruit walls. The fruit wall is composed of a uniseriate inner palisade layer and an outer pericarp (Fig. 6M). The palisade layer comprises individual cells that are radially elongate with a prominent thickened cell wall (Fig. 6G) and range in size from 12–15 µm long x 5–7 µm wide. Surface features of the inner carpel wall show short, rectangular cells, with oblique end walls (Fig. 7E, F). Cells are slightly raised above the general surface (Fig. 7J).
Seeds are 8.0 mm long x 2.3 mm wide (L/W ratio, 3.4 :1), elongate, and have a relatively long distal wing (Figs. 7F, 8D). Cells of the seed coat are tangentially elongate, rectangular to polygonal with thickened anticlinal walls and mostly straight end walls (Fig. 8H). The epidermal cells of the seed coat are cuboidal in cross-section and are covered by a thin cuticle (Fig. 9H). Below the epidermis are 2–3 rows of small parenchyma cells followed by 2–3 rows of macrosclereids, each with a small lumen (Fig. 9H). To the inside of this fibrous layer are 2–3 rows of crushed epidermal cells of the outer integument, followed by parenchymatous cells of the endosperm (Fig. 9H).
Characteristic features of this species include the braided appearance of extrafloral processes, the broad style bases (Fig. 6C), the elongate seeds with a prominent distal wing, and the thickened cell walls of the inner carpel wall.
Phylogenetic analysis
Morphological cladistic analysis of Liquidambar and its close extant and fossil relatives with Exbucklandia and Hamamelis as outgroups revealed two MPT of 57 steps with a consistency index (CI) of 0.89, a retention index (RI) of 0.86, and a rescaled consistency index (RC) of 0.79 (Fig. 10A). The two trees differed in the position of the fossil Liquidambar changii. It was either sister to the extant Liquidambar clade (Fig. 10A) or formed a trichotomy with L. acalycina and the subclade of L. formosana, L. orientalis, and L. styraciflua (not shown). Of 42 character-state changes detected, 32 were parsimony informative. The family Altingiaceae is supported as monophyletic with the Cretaceous fossil from eastern North America, Microaltingia apocarpela, appearing as sister to the rest of the family. Altingia is sister to Liquidambar, which was also supported to be monophyletic by the morphological data. Within Liquidambar, the Miocene fossil taxon from western North America, L. changii, is cladistically basal. Liquidambar acalycina forms a clade with L. formosana and the subclade of L. orientalis and L. styraciflua.
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Both the partition-homogeneity test and the Templeton's significantly less parsimonious test (SLPT) indicated that the morphological and the molecular matK data sets were not congruent (P = 0.01 and P = 0.0023, respectively). The conflicts between the trees of the two datasets (Fig. 10A, B) lay in the positions of Altingia chinensis, L. acalycina, and L. formosana. The morphological cladistic analysis supports the monophyly of Liquidambar. However, the molecular analysis suggests that Altingia was derived from within Liquidambar (Fig. 10B); specifically, it is sister to the L. acalycina–L. formosana clade.
The tree from the analysis constraining the monophyly of Liquidambar acalycina and L. formosana was four steps longer as compared to the unconstrained tree, with a CI = 0.83 and a RI = 0.75. When the morphological data was constrained against the matK phylogeny, excluding the two fossil taxa Microaltingia apocarpela and L. changii, the tree was nine steps longer, with a CI = 0.77 and a RI = 0.63. Constraining the three eastern Asian species (Altingia chinensis, L. acalycina, and L. formosana) as a clade, the tree was six steps longer with a CI = 0.81 and RI = 0.70.
DISCUSSION
Based on the comparative study of infructescence anatomy and morphology in Liquidambar, we can now identify several characters that appear to be both consistently reliable and taxonomically informative. They include some of the traditionally recognized characters, defined in more detail, as well as several new characters of anatomy and micromorphology. Among those we will discuss are (1) overall construction of infructescence; (2) infructescence size, shape, fruit number, and details of axis; (3) presence and type of extrafloral processes; (4) style morphology; (5) pericarp anatomy; (6) carpel surface micromorphology; (7) seed morphology; (8) seed anatomy; and (9) seed surface micromorphology.
Overall construction and structural integrity of infructescence
Infructescences vary in the degree to which fruits are attached to one another. In L. styraciflua, L. orientalis, and L. formosana, fruits are tightly attached: in L. acalycina, as in Altingia, fruits are loosely attached and the infructescence falls apart upon sectioning (K. Pigg, S. Ickert-Bond, personal observation). A similar organization occurs in the altingioid fossil infructescence Steinhauera Presl, which occurs in many European localities in the Eocene (Mai, 1968
).
Infructescence size, shape, fruit number and details of axis
The number of fruits per infructescence varies with L. orientalis having the largest number (48), L. styraciflua and L. formosana with fewer (37 and 34, respectively), and L. acalycina with the fewest (26). The relative size of infructescences also varies, with the largest infructescences found in L. styraciflua (30 mm long), those of intermediate size in both L. acalycina and L. formosana (27 mm long), and those of L. orientalis, the smallest, at only 15 mm. Width varies accordingly within individual measurements (Table 2), and comparable relative dimensions are found in carpel size. In our observation it is interesting to note that L. orientalis has a much smaller infructescence with considerably more fruits than the other species of Liquidambar. When scaled to the same infructescence size, L. orientalis has twice as many fruits as L. formosana, three times as many fruits as L. acalycina, and 2.4 as many fruits as L. styraciflua. Although all four species have spherical infructescences, L. orientalis is somewhat wider than long, with a L/W ratio of 0.9 : 1. In comparison to these extant species, the Cretaceous fossil, Microaltingia, is tiny (7 mm in diameter) with 8–12 fruits, and the Miocene L. changii around 2.5 cm in diameter with approximately 25–30 fruits per head (Zhou et al., 2001
; Pigg et al., 2004
).
Presence and type of extrafloral processes
The presence or absence of extrafloral processes has been historically important in the delimitation of species of Liquidambar (Harms, 1930
). In our study we observed that all species of Liquidambar, as well as several representative species of Altingia, typically have a group of 8–12 extrafloral structures of some sort surrounding each mature fruit. The presence of these types of structures throughout Altingiaceae was noted previously (Bogle, 1986
). The most obvious of these are the "extra spines" of L. formosana (Fig. 4C); however, spinose structures also occur in L. acalycina (Fig. 3B). Braided knob-like structures as well as occasional short spinose protuberances occur in L. styraciflua (Fig. 6A, C), and broad, slightly raised "smooth" areas occur in L. orientalis (Fig. 5A). In Altingia, structures found in the same positions are mostly small tubercles (S. Ickert-Bond, unpublished data).
These extrafloral processes have variously been described by the neutral term phyllome (sensu Esau, 1965
) by Bogle (1986)
or interpreted as (1) bracteoles (Guillaumin, 1920
), (2) staminodia (Tong, 1930
), (3) calyx lobes (Chang, 1962
, 1973
), (4) papillae on the surfaces of the capsules (Schmitt, 1965
), or (5) as vestigial styles of sterile flowers interspersed among fertile flowers (Harms, 1930
). In their ontogenetic study, Wisniewski and Bogle (1982)
observed that the extrafloral processes developed late in L. styraciflua, well after stamens and functional carpels were initiated. However, their observations do not resolve the homology of these structures.
To determine the homologies of these extrafloral processes, we considered the morphology, position, and merosity of the five types of structures previously proposed as homologous by various authors. If, for example, the extrafloral structures are homologous with (1) bracteoles or modified leaves associated with the inflorescence (Gifford and Foster, 1989
), one would expect only a single pair of these structures to occurr alternately at the base of each gynoecium, as in the closely related hamamelid clade, and that they would most likely appear leafy. If they are homologous with (2) staminodia, transitional forms leading to stamens would be expected (Bogle, 1986
). Within the closely related Hamamelidaceae, rare reports of sterile phyllomes transitional to stamens are known from Corylopsis Siebold et Zucc. (Tong, 1930
; Endress, 1967
) and Loropetalum R. Br. (Mione and Bogle, 1990
). If the structures are homologous to (3) calyx lobes, they would quite likely conform to the merosity of the floral parts within the closely related hamamelids. Among hamamelids that develop a perianth, the calyx is four to five-merous; however, in those lacking a well-developed corolla, the organ merosity is not fixed (Endress, 1989c
). The structures are unlikely to be homologous with (4) papillae, as they are vascularized, woody, and composed of multiple layers of cells.
Of these several alternatives, the most probable explanation to us is that the extrafloral structures are homologous to (5), a group of nondeveloping flowers that surround a viable flower. Our interpretation is supported by the similar morphology of these structures, particularly those of L. acalycina and L. formosana, with viable fruits, and their often paired appearance, as would be expected if these structures are derived in the same way as the bilocular fruits of Altingiaceae.
To better understand whether this interpretation might be valid, we made small clay models of normal-sized and smaller bilocular fruits and positioned them together on a branching system (Fig. 2D). The resulting model has 8–10 small "extrafloral processes" surrounding a "viable flower." The actual number of surrounding processes would be somewhat variable, because of the small amount of torque expected in the tightly packed, helically arranged fruits. Despite their superficial differences, the well-developed spinose extrafloral structures of L. acalycina and L. formosana, the ornamented "braided" peripheral rims of L. styraciflua, and the broad areas between fruits of L. orientalis are found in the same relative positions, supporting a positional criterion of homology (e.g., Kaplan, 1984
).
Styles in inflorescences and infructescences
Persistent styles are typically characteristic of mature infructescences of Liquidambar (Bogle, 1986
; Ferguson, 1989
); however, information on the morphology of styles within inflorescences at anthesis is mostly lacking (Tardieu-Blot, 1965
). This character was studied in greater detail. Styles within inflorescences are fleshy and thick in L. styraciflua and L. orientalis, and considerably narrower in L. formosana. The stigmatic surfaces in all species are relatively broad and decurrent (Bogle, 1986
; Endress and Igersheim, 1999
). In L. styraciflua and L. orientalis, the fleshy stigmas end in a short, curving tip upon which the stigmatic surface occurs. In contrast, the styles of L. formosana are distinctly coiled. Fresh inflorescences of L. acalycina were not available for study; however, from examination of inflorescences on herbarium sheets the styles in L. acalycina appear narrow. The stigmatic surfaces of Liquidambar are unusual in having unicellular papillate epidermal cells superimposed on multicellular protuberances, while the stigmatic surfaces of the Hamamelidaceae are generally described as mostly not papillate, but with pluricellular warts (Endress, 1989b
, 1993
; Endress and Igersheim, 1999
). As the infructescences develop, styles of all species tend to dry out and become woody and brittle. The stigmatic surfaces often break off or abrade at this stage; however, the styles and their triangular bases typically persist (Figs. 3A, 3B, 4A, 5E, 6A, 6C). With desiccation the styles and style bases become vertically striate (Figs. 3B, 4C, 6C).
Carpel/locule shape
All fruits of Liquidambar are spherical infructescences with numerous bilocular fruits packed into a sphere. There is variation in the shape of carpels. Liquidambar formosana and L. styraciflua have elongate locules (Figs. 4C, 4E, 6H, 6I), those of L. orientalis are obovate (Fig. 5D), and those of L. acalycina are broadly obovate (Fig. 3C). Those of Microaltingia and L. changii can be compared most favorably with those of L. acalycina (Zhou et al., 2001
; Pigg et al., 2004
).
Pericarp anatomy
We demonstrate for the first time that characters of pericarp anatomy vary among different species of Liquidambar. In particular, the innermost layer of palisade cells that lines each locule varies in cell number and anatomy. It is a single cell thick in L. styraciflua (Fig. 6E), L. orientalis (Fig. 5M), and L. formosana (Fig. 4H) and two cells thick in L. acalycina (Fig. 3H). Among species with a single palisade, L. formosana has cells with thin walls (Fig. 4H), while in L. styraciflua (Fig. 6E) and L. orientalis (Fig. 5M), cells have a prominent thickening on the inner tangential wall. In contrast, organization of the inner part of the outer pericarp is similar in all species, with fragments of the innermost tangentially oriented cell layers typically adhering to the inner palisade layer. The ground tissues of mature infructescences are often difficult to characterize because they are usually ruptured and torn along planes of weakness, particularly within the area inside of the vascular strands (Fig. 2E). To the outside, the vascular bundles among species vary further with the presence or absence of druses, differences in number and distribution of resin canals, and amounts and type of sclerenchyma (Figs. 3F, 3J, 4F, 4K, 4L, 5K, 5L). The epidermis also varies among species, with L. acalycina and L. styraciflua (Fig. 4K) characterized by tabular cells, L. orientalis by papillate types (Fig. 5L), and L. formosana by radially elongate cells.
Carpel surface micromorphology
The innermost carpel wall appears as a distinct lustrous layer during hand-sectioning of infructescences in Liquidambar (Figs. 4E, 7C) and separates readily from the remaining pericarp. Surface micromorphology has a fairly homogenous cell pattern with short rectangular cells, that have oblique end walls in L. formosana (Fig. 7H), L. orientalis (Fig. 7I), and L. styraciflua (Fig. 7J). In contrast, L. acalycina has tangentially elongate cells that appear rod-like and anastomose (Fig. 7G).
Seed morphology
Whereas the large majority of genera in the closely related family Hamamelidaceae s.s. have ballistic seeds lacking a wing, those of the Altingiaceae have either distally winged or circularly flanged seeds (Tiffney, 1986
; Fig. 2H). Wing morphology ranges from large distal wings in L. styraciflua (Fig. 6F) and L. formosana (Fig. 4M) to somewhat shorter wings in L. orientalis (Fig. 5C), while L. acalycina has small circular to triangular wings that are similar to those of both Semiliquidambar and Altingia (Fig. 3K, L; S. Ickert-Bond, unpublished data). Seeds of the fossil L. changii have circular flanges (Pigg et al., 2004
), while those of Microaltingia are reported to lack wings (Zhou et al., 2001
). Seeds in Altingiaceae are typically speckled or striped, but variation in this feature, even within a single infructescence, does not allow for species delineation based on this character.
Seed anatomy
Another feature that has been described previously for some taxa formerly recognized within Hamamelidaceae (including Liquidambar) is seed anatomy (Shoemaker, 1905
; Melikian, 1971
, 1973
; Rao, 1974
; Zhang and Wen, 1996
). In Liquidambar, the outer epidermis of the inner integument constitutes most of the seed coat, in contrast to most hamamelidaceous seeds, in which the tegmen does not contribute much to the seed coat (Rao, 1974
). The seed coat in Liquidambar is well differentiated. A uniseriate epidermis is covered by a cuticle on upper anticlinal walls in L. formosana (Figs. 4J, 8J), while in both L. styraciflua and L. orientalis, the cuticle is found on both anticlinal and periclinal walls (Fig. 8I; Melikian, 1973
). A uniseriate hypodermis contains druse crystals in L. styraciflua (Fig. 8I, K), L. formosana (Fig. 4J), and L. orientalis, while we failed to find them in L. acalycina. To the inside of the hypodermis are 1–3 layers of macrosclereids. Both L. orientalis and L. styraciflua have macrosclereids in 1–2 rows, while in L. formosana there are 2– 3 rows. In a detailed study of seed coats in Hamamelidaceae, Melikian (1973)
concludes that the very thin seed coats of Liquidambar and Altingia are specialized in comparison to the relatively thick seed coats in the rest of the Hamamelidaceae s.s., which was considered "primitive."
Seed surface micromorphology
Overall seed surface micromorphology in Liquidambar is relatively homogeneous with cells arranged parallel to the long axis of the seed (Fig. 8E–H). The surface of seed coats in L. acalycina differs from the other three species in having polygonal (4–5 sided) cells that are slightly elongate and appear slightly thickened on the anticlinal walls (Fig. 8E), while the other taxa have tangentially elongated and rectangular cells with thickened anticlinal walls (Fig. 8F). Among the three species with rectangular cells of the seed coat, L. styraciflua (Fig. 8H) and L. formosana (Fig. 8F) have straight end walls, while L. orientalis has oblique end walls (Fig. 8G).
Evolution of fruit dispersal in Altingiaceae
Although the genera comprising the Altingiaceae have been recognized as closely related to those members of the Hamamelidaceae s.s., there are several important differences between these two groups that have, in part, led to the recognition of the Altingiaceae as a distinct family (Judd et al., 2002
). The Altingiaceae are distinguished from the Hamamelidaceae s.s. by the presence of polyporate rather than tricolpate pollen. They also have multiseeded, bilocular carpels, that lack an elaborate dispersal mechanism, in contrast to the single-seeded capsules of the Hamamelidoideae of Hamamelidaceae, which have an explosive (ballistic) ejection mechanism (Tiffney, 1986
; Endress, 1993
). Exbucklandia (Exbucklandioideae) and Rhodoleia (Rhodoleioideae) also lack this explosive dispersal syndrome (Endress, 1989b
), suggesting that the ballistic dispersal is perhaps a synapomorphy for subfamily Hamamelidoideae.
Within Liquidambar, L. formosana and L. styraciflua have elongate locules (Figs. 4C, 4E, 6H, 6I), those of L. orientalis are obovate (Fig. 5D), and those of L. acalycina are broadly obovate (Fig. 3C). There is a correlation between seed shape and locule shape, with seeds with elongate distal wings in L. formosana and L. styraciflua, and those with a shorter distal wing in L. orientalis. Liquidambar acalycina seeds have a circular to triangular flange encircling the seed body, and its locules are considerably shorter and broader. Carpel surface micromorphology correlates with this as well, as does the overall construction and structural integrity of infructescences. It is interesting that while the first three species have tightly interconnected infructescences, those of L. acalycina are only loosely attached to one another. The fossil L. changii has locules and seeds with shapes similar to those of L. acalycina (Pigg et al., 2004
).
In comparison to the hamamelids with a ballistic or explosive dispersal mechanism, the composition and structure of infructescences of the Altingiaceae with 1-several seeded fruits, crowded and partially immersed in an infructescence, functionally constrains these taxa to a nonballistic dispersal mechanism (Endress, 1989c
). Within the family, several variations of dispersal have evolved. Whereas in general, the more elongate seeds of Liquidambar are abiotically dispersed, Vink (1957)
reports monkeys, birds, and ants feeding on the shorter, fatter seeds of the closely related Altingia excelsa Nor., which Kalshoven (1937)
describes as containing oil.
Differences in seed and locule shape among species of Liquidambar seem also to play a role in dispersal, because the broader seeds of L. acalycina and the fossil L. changii would have to have a broader opening during dehiscence as compared to the slender, elongated seeds of the other three extant species of Liquidambar. Many wind-blown infructescences contain the large viable seeds inside, while only the smaller aborted seeds have been displaced from within the pendulous infructescences.
Morphological evolution of Liquidambar
The morphological cladistic analysis supports the monophyly of Liquidambar. However, the molecular analysis suggests that Altingia is nested within Liquidambar (Fig. 10B); specifically, it is sister to the L. acalycina–L. formosana clade. Morphological characters that differentiate Liquidambar from Altingia include size and shape of inflorescence, number of florets (and fruits) per head, presence or absence of spines in the infructescence, persistence of styles on infructescence, and mode of fruit dehiscence (Ferguson, 1989
; Zhang et al., 2003
). Semiliquidambar has morphology intermediate between the two other genera and is thought by some to be an intergeneric hybr
