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Ann Bot. 2009 November; 104(6): 1243–1253.
Published online 2009 September 16. doi:  10.1093/aob/mcp232
PMCID: PMC2766214

Early reproductive developmental anatomy in Decaisnea (Lardizabalaceae) and its systematic implications

Abstract

Background and Aims

Decaisnea insignis, known as ‘dead man's fingers’ (Lardizabalaceae), is widely distributed in China and the Himalayan foothill countries. This economically important plant, which is the only species in the genus, has not been the subject of any embryological studies aside from one brief, older paper that lacks micrographs. Data on Decaisnea are also important because its systematic position has been unstable since the genus was established in 1855. Therefore, the objectives of this study were: (a) to use modern microscopy to document early reproductive anatomical development in Decaisnea; and (b) to compare qualitatively these early embryological characters with allied taxa in a systematic context.

Methods

Decaisnea insignis floral buds and inflorescences were regularly collected from Shaanxi Province, China and prepared for light microscopy. The embryological characters studied were qualitatively compared with those of allied taxa via a thorough examination of the existing literature.

Key Results

Early reproductive anatomy in Decaisnea was documented and novel revelations made. It was discovered that the pollen is shed when three-celled (not two-celled, as previously reported), and that endosperm formation is nuclear (not cellular or helobial, as previously reported). These two newly revealed embryological characters are not found in any other members of Lardizabalaceae. Furthermore, neither are persistent antipodal cells, which we confirmed to be present in Decaisnea.

Conclusions

Decaisnea and other Lardizabalaceae characteristically have tetrasporangiate anthers, a secretory tapetum, simultaneous microsporocyte cytokinesis, primarily bitegmic, crassinucellate ovules, and a Polygonum type embryo sac. However, in the family, persistent antipodals, nuclear endosperm, and pollen shed at the three-celled stage are only found in Decaisnea. These embryological data prompted the suggestion that Decaisnea needs elevation above the level of genus.

Keywords: Decaisnea insignis, embryology, endosperm, Lardizabalaceae, microscopy, pollen, reproductive anatomy, systematics

INTRODUCTION

Decaisnea is a monotypic genus of Lardizabalaceae, with the species, Decaisnea insignis (Griffith) Hook. f. & Thomson (Chen and Tatemi, 2001), widely distributed from central to south-western China, extending into Bhutan, Myanmar, Nepal, Sikkim and north-eastern India. The plant is nicknamed ‘dead man's fingers’, as it possesses racemes of striking deep purplish-blue elongated fruits (follicles). The plant is economically important, as it is readily cultivated as an ornamental, and its fruits are deemed to be a delicacy. However, in spite of the value of Decaisnea, it has not been the subject of any dedicated embryological studies aside from a solitary and brief older paper by Swamy (1953), which is limited in that it exclusively presents line drawings. As such, a modern examination of the embryology of Decaisnea is needed.

The systematic position of Decaisnea has been unstable since the genus was established in 1855 (Griffith, 1855). Presently, the Lardizabalaceae is generally recognized to have nine genera: Akebia, Boquila, Decaisnea, Holboellia, Lardizabala, Parvatia, Sargentodoxa, Sinofranchetia and Stauntonia (Qin, 1997; Chen and Tatemi, 2001). However, Loconte and Estes (1989) state that Decaisea could be treated as a subfamily named Decaisneoideae within Lardizabalaceae, as indicated by an outgroup comparison and parsimony analysis of 34 genera within Ranunculales. Further to this, Loconte et al. (1995) conclude that the genus Decaisnea should be a new family within a new order Lardizabalales within ranunculids as implied by a morphologically based cladistic analysis on 116 ingroup taxa and five outgroups coded for 109 characters and 192 apomorphic character states. Thorne (2000, 2007) acknowledges the elevated status of Decaisnea suggested by Loconte and Estes (1989) and Loconte et al. (1995).

Qin (1989, 1997), however, regards Decaisnea as the only genus within the tribe of Decaisneeae, which, along with three other tribes (Sinofranchetieae, Lardizabaleae and Akebieae) comprise Lardizabalaceae. Many authors support Qin's taxonomic treatment (Qin, 1989). Chen and Tatemi (2001) agree that Decaisnea is a genus within Lardizabalaceae; this view is supported by the Angiosperm Phylogeny Group (APG II, 2003) and Mabberly (2008). Using a cladistic analysis of 43 morphological characters sensu lato, Wang et al. (2002) provide support for Qin's view (Qin, 1989), but note that the phylogeny of Lardizabalaceae needs further study. Zhang et al. (2005) also agree with Qin's taxonomic system (Qin, 1989). Hoot et al. (1995a, b, 1999) constructed a molecular phylogeny based on chloroplast and nuclear DNA sequences, which resolved Decaisnea as a member of Lardizabalaceae, but did not include morphological or developmental characters.

Embryological data have been helpful in inferring relationships among genera and families (Bhojwani and Bhatnagar, 1978). Endress and Igersheim (1999) present an excellent review of gynoecium diversity and systematics of the basal dicots, which includes a useful synopsis of ovular characteristics of the Lardizabalaceae, but does not specifically describe embryology nor focus on the genus Decaisnea. As such, a study of embryological ontogeny in Decaisnea would not only contribute new information regarding reproductive development in this important plant, but would also provide a new suite of characters for systematic studies; Swamy (1953) did not compare his limited embryological data on Decaisnea with those of allied taxa nor discuss their systematic implications. Here, anther, pollen, ovule, embryo sac and early endosperm development in Decaisnea insignis are studied. These early embryological characters are then compared with allied taxa and the systematic implications discussed.

MATERIALS AND METHODS

Collection and photography of whole floral buds and flowers

About five floral buds or maturing inflorescences of Decaisnea insignis (Griffith) Hook. f. & Thomson were collected every 5 or 6 d from Taibai Mountain in Shaanxi Province, China (altitude 1200–1500 m; voucher: Zhang 20030609, SANU) from 1 March 2005 to 1 May 2006 in order to obtain a range of developmental stages. Samples were fixed in formalin–acetic acid–ethanol (FAA) 2 : 2 : 1 volume/volume (v/v/v). Floral buds and flowers (about three per sampling date) were dissected and photographed using a Nikon DXM 1200 stereo microscope.

Preparation for sectioning and microscopy

Fixed male and female flower buds were dehydrated in an ethanol series (70 %, 85 %, 95 %, 100 % and 100 % ethanol, 2 h each), and embedded in paraffin wax. Approximately ten serial sections of individual flowers (at least two flowers) were cut at 6–9 µm, stained for 4 h in 4 % Heidenhain's iron-alum, washed for 40 min with H2O, stained for 4 h with 0·05 % hematoxylin, washed again with H2O (30 min), and mounted on slides in a gelatin solution (1 g gelatin, 100 mL H2O, 2 g phenol, 15 mL glycerol; Li, 1978). Photographs were taken with an Olympus SP-565UZ digital camera mounted on an Olympus BH-2 photomicroscope equipped with Nomarski optics. The tonal qualities of the images were adjusted, labels were added, and plates assembled with Adobe Photoshop CS2 and CS3.

RESULTS

Floral bud morphology

Decaisnea insignis plants used in this study were found to be polygamo-monoecious, as individuals possessed male flowers, female flowers and bisexual flowers; the strictly monoecious condition is apparently rare. For consistency, only unisexual flowers were examined. In early spring, winter buds are ovoid and possess two outer scales (Fig. 1A). The inflorescence is a terminal panicle of racemes (Fig. 1B). Each flower is subtended by a bract (Fig. 1C). Each flower has six subimbricate sepals and lacks petals. Male flowers have six stamens with oblong anthers, and three carpellodes that are small and concealed within the filament tube (Fig. 1D). Female flowers have staminodes that are either free, or, less frequently, connate at the base, and have three cyclically arranged straight carpels (not shown). The mature fruit consists of three follicles.

Fig. 1
Inflorescence and floral structure of Decaisnea insignis. (A) Young leaves surrounding an immature inflorescence. One of two outer scales (sc) is visible. (B) Inflorescence branches emanate from the base of the inflorescence axis. The two lowermost branches ...

Development of the anther wall

The anther is tetrasporangiate (Fig. 2A). The single-celled archesporium is hypodermal and undergoes a periclinal division, resulting in a primary parietal layer and a primary sporogenous layer. The parietal layer divides periclinally to form two layers: the inner layer contributes to the tapetum (Fig. 2B), while the outer parietal layer undergoes another periclinal division, resulting in an endothecium toward the outside and a middle layer toward the inside (Fig. 2C). The mature anther wall is thus comprised of five or six layers: a single-layered epidermis, a single-layered endothecium, two or three middle layers, and a single-layered tapetum (Fig. 2D), and conforms to the dicotyledonous form of anther wall development.

Fig. 2
Development of the anther wall and sporogenous tissue. (A) Cross-section through the six tetrasporangiate anthers (an); arrowheads indicate the four sporangia of a representative anther. c, Carpellode; se, sepal. (B) Periclinal division of the hypodermal ...

The tapetum is of dual origin: most of it develops from the primary parietal layer, but a component also arises from the ground tissue of the connective side (not shown). After formation of the anther wall but before microsporogenesis, about half of the tapetal cells undergo mitosis without cytokinesis, becoming binucleate (Fig. 3A). Upon initiation of microsporocyte meiosis, the tapetal cells elongate radially and protrude into the anther locule (Fig. 3B). The tapetal cells degenerate at their original sites following microsporogenesis. Therefore, the tapetum is of the secretory (glandular) type. The epidermis persists at maturity, the endothecium develops fibrous thickenings, and the middle layers are ephemeral, degenerating shortly before the microspores develop into pollen grains.

Fig. 3
Maturation of the tapetum is concurrent with microsporogenesis and microgametogenesis in Decaisnea insignis. (A) Prior to microsporogenesis, about half of the tapetal cells (t) undergo mitosis without cytokinesis, becoming binucleate (arrowheads). Microsporocytes ...

Microsporogenesis and microgametogenesis

After formation of the anther wall, each microsporangium contains numerous sporogenous cells (Fig. 2D). Microsporocytes originate from the primary sporogenous layer as well as from secondary sporogenous cells (Fig. 3A). Individual microsporocytes become enclosed in a thick callose wall when their nuclei enter prophase of meiosis I (Fig. 3B). Meiosis II is followed by simultaneous cytokinesis with centripetally advancing constriction furrows, and results in tetrahedral microspore tetrads, which enlarge and acquire thick callose walls (Fig. 3C). Shortly after callose formation, the callose walls promptly break down, releasing microspores from the tetrad (Fig. 3D). Then, formation of a large vacuole pushes the single nucleus of each freed microspore toward the microspore wall (Fig. 3E). Each microspore divides to form a large tube (vegetative) cell in the vicinity of the large vacuole and a small generative cell in the region that housed the original wallward microspore nucleus (Fig. 3F). The generative cell undergoes a further division, resulting in two sperm cells (Fig. 3G). Tricolpate pollen grains are shed at this three-celled stage (Fig 3H).

Megasporogenesis and megagametogenesis

Numerous ovules are found in two rows on either side of an adaxial carpel suture (Fig. 4A). The archesporium is one-celled (Fig. 4B), and transforms into a megasporocyte by cutting off a parietal cell (Fig. 4C). The parietal cell undergoes further divisions to form the nucellar tissue (Fig. 4D), which causes the megasporocyte to become deep-seated within the ovule. Thus, the ovule is crassinucellate.

Fig. 4
Megasporogenesis in Decaisnea insignis. (A) Numerous ovules (ov) are found in two rows on either side of an adaxial carpel suture. fu, Funiculus; pl, placenta. (B) The archesporium (arrowhead) is one-celled and situated in a hypodermal position. (C) The ...

Following parietal cell divisions, the megasporocyte undergoes meiosis I, resulting in a dyad (Fig. 4D). Meiosis II produces a T-shaped tetrad of megaspores (Fig. 4E). The three micropylar megaspores of the tetrad degenerate, while the chalazal megaspore becomes visibly functional, possessing a prominent nucleus (Fig. 5A). The functional megaspore develops successively into a two-nucleate (Fig. 5B), four-nucleate (Fig. 5C) and, finally, eight-nucleate embryo sac (Fig. 5D, E) by three mitotic divisions. Thus, the mode of embryo sac formation is of the Polygonum type.

Fig. 5
Megagametogenesis in Decaisnea insignis. (A) The three micropylar megaspores of the tetrad have become degenerated megaspores (dm), while the chalazal megaspore is obviously the functional megaspore (fm), as it possesses a prominent nucleus (arrow). (B) ...

The three micropylar nuclei become the egg and two synergids, collectively comprising the egg apparatus (Fig. 5D). The unfertilized egg cell is highly vacuolate (Fig. 5D). The two median nuclei become the polar nuclei (Fig. 5D, E), and the chalazal nuclei become the three antipodals (Fig. 5D, E). The polar nuclei fuse in the centre, forming the fusion (secondary) nucleus of the central cell and remain in this central location until fertilization (Fig. 6A).

Fig. 6
Events immediately surrounding double fertilization. (A) The two polar nuclei of the central cell fuse to form the fusion nucleus (fn) immediately prior to fertilization. The fusion nucleus resides in a central position until fertilization. (B) Zygote ...

Double fertilization and development of endosperm

Following fertilization of the egg cell, the zygote becomes densely cytoplasmic (Fig. 6B). Soon after, the primary endosperm nucleus of the central cell migrates toward the antipodals, which persist at the chalazal pole of the embryo sac (Fig. 6C). The first division of the primary endosperm nucleus is not accompanied by wall formation (Fig. 6D). Free nuclear endosperm formation ensues within several mitotic divisions (Fig. 6E). As early nuclear endosperm develops, the two synergids become reduced, while the antipodals persist at the chalazal pole (Fig. 6E).

Ovule development

Ovule development is concurrent with events of megasporogenesis and megagametogenesis. The ovule is nearly straight early in development (Fig. 7A), but a slight curvature becomes noticeable when the megasporocyte has completed meiosis II (Fig. 7B), and as the ovule approaches maturity, it gradually becomes anatropous (Fig. 7C), completing curvature when the embryo sac reaches the eight-nucleate stage (Fig. 7D). The ovule is bitegmic. The inner integument is initiated simultaneously with the onset of megasporogenesis (Fig. 7A), while the outer integument is initiated when the embryo sac has become two-nucleate. The integuments do not complete development until the embryo sac reaches the eight-nucleate stage, when the endostomic micropyle becomes evident (Fig. 7D).

Fig. 7
Ovule development in Decaisnea insignis. (A) The ovule is nearly straight early in development. The inner integument (i) is initiated simultaneously with the onset of megasporogenesis. fu, Funiculus. The megasporocyte is entering meiosis, and can be better ...

DISCUSSION

Early embryological features of Decaisnea and other Lardizabalaceae

Development of the anther wall

It has been found that development of the anther wall is of the dicotyledonous type in Decaisnea, since the endothecium and middle layers originate from a single layer of cells, and the tapetum, which is secretory rather than amoeboid, does not originate from connective tissue. Most of the present observations on anther development confirm and expand those of Swamy (1953), as wall layer ontogeny is explicitly documented. However, while Swamy (1953) suggests that all tapetal cells are binucleate, it is noted that some remain uninucleate, even late in development.

There are noteworthy differences in early embryological development of Decaisnea when compared with other genera in the Lardizabalaceae. Development of the anther wall is of the basic type in Sargentodoxa (Liu and Sheng, 2003), and of both the dicotyledonous and basic type in Sinofranchetia (Zhang et al., 2005). Like Decaisnea, the tapetum is secretory in Holboellia (Bhatnagar, 1965), Sargentodoxa (Liu and Sheng, 2003) and Sinofranchetia (Zhang et al., 2005). In Stauntonia hexaphylla, the tapetal cell walls appear to break down (Yoshida and Nakajima, 1978), as is typical of an amoeboid tapetum, but the protoplasts remain in situ, which is characteristic of a secretory tapetum. The tapetal cells typically contain no more than two nuclei in Decaisnea and Holboellia angustifolia (Wang, 2001), while they contain two to four nuclei in Holboellia latifolia (Bhatnagar, 1965).

Microsporogenesis and microgametogenesis

It has been observed that cytokinesis following microsporocyte meiosis is simultaneous, and results in tetrahedral tetrads in Decaisnea. The same is true for Holboellia angustifolia (Wang, 2001), Sargentodoxa (Liu and Sheng, 2003) and Sinofranchetia (Zhang et al., 2005). However, tetrads are tetrahedral or decussate in Holboellia latifolia (Bhatnagar, 1965). It has been found that the mature tricolpate pollen grains of Decaisnea are three-celled at the time of shedding, and not two-celled as reported by Swamy (1953). Holboellia latifolia (Bhatnagar, 1965), Holboellia angustifolia (Wang, 2001), Sargentodoxa (Liu and Sheng, 2003) and Sinofranchetia (Zhang et al., 2005) are also reported to have two-celled mature pollen grains when shed.

Megasporogenesis and megagametogenesis

The present results indicate that Decaisnea possesses T-shaped megaspore tetrads. Holboellia latifolia also possesses T-shaped megaspore tetrads (Bhatnagar, 1965), as is found in Sargentodoxa, although the megaspore tetrads in this genus are occasionally linear (Sheng et al., 2005). The megaspore tetrads are linear in Holboellia angustifolia (Wang, 2001) and Sinofranchetia (Zhang et al., 2005). Embryo sac development in Decaisnea conforms to the Polygonum type; this is also the case in Holboellia latifolia (Bhatnagar, 1965), Akebia spp. (Yoshida and Michikawa, 1973), Stauntonia hexaphylla (Yoshida and Nakajima, 1978), Holboellia angustifolia (Wang, 2001), Sargentodoxa (Sheng et al., 2005) and Sinofranchetia (Zhang et al., 2005). The present findings show that the antipodals in D. insignis are persistent and large; these findings are consistent with Swamy's report (Swamy, 1953). In contrast, the antipodals are small and ephemeral in Akebia spp. (Yoshida and Michikawa, 1973), Holboellia latifolia (Bhatnagar, 1965) and Stauntonia hexaphylla (Yoshida and Nakajima, 1978). In Decaisnea, the polar nuclei fuse before fertilization. While Swamy (1953) similarly notes that the two polar nuclei of Decaisnea fuse, his report suggests that the unfertilized fusion nucleus migrates toward the antipodals. We maintain that migration toward the antipodals only occurs after double fertilization has taken place, as the large nucleus of the central cell is only found near the antipodals following a visible change in the egg cell to a more cytoplasmic state, indicative of fertilization (Raghavan, 1998).

Double fertilization and development of endosperm

The present findings regarding early endosperm development in Decaisnea do differ somewhat from those of Swamy (1953). Firstly, as mentioned, it is clear that it is the fertilized primary endosperm nucleus that migrates toward the antipodals, not the unfertilized fusion nucleus. Furthermore, we believe that endosperm development in Decaisnea is of the free nuclear type, not the helobial type as described by Swamy (1953). Using line drawings, Swamy (1953) documents a first cellular division of the primary endosperm nucleus that partitions the endosperm into a micropylar and chalazal chamber; while Swamy (1953) did not explicitly use the term ‘helobial’, he had effectively described such a type of endosperm development (Raghavan, 1998). However, no evidence of such a division has been seen, and we maintain that the endosperm in Decaisnea is of the free nuclear type. All members of the Lardizabalaceae are generally believed to have ab initio cellular endosperm (Johri et al., 1992).

Ovule development

In Decaisnea, ovules are anatropous, bitegmic and crassinucellate (see also Endress and Igersheim, 1999). However, we disagree with Swamy's (1953) report regarding the timing of integumentary development. In the present study it was found that the inner integument is initiated simultaneously with the onset of megasporogenesis, the outer integument is initiated when the embryo sac has become two-nucleate, and that the integuments do not complete development until the embryo sac reaches the eight-nucleate stage. Swamy (1953), on the other hand, suggests both integuments are initiated simultaneously, and that integumentary development is complete at the four-nucleate stage. Akebia quinata (Endress and Igersheim, 1999), Holboellia angustifolia (Wang, 2001) and Sinofranchetia (Endress and Igersheim, 1999; Zhang et al., 2005) also have ovules that are anatropous, bitegmic and crassinucellate. The ovules are hemianatropous in Sargentodoxa (Sheng et al., 2005).

Embryological comparison of Decaisnea with allied groups: systematic implications

The present study shows that Decaisnea and other genera of Lardizabalaceae generally share the following embryological characters: tetrasporangiate anthers (Johri et al., 1992), secretory tapetum (Bhatnagar, 1965; Liu and Sheng, 2003; Zhang et al., 2005), simultaneous cytokinesis in the microsporocytes (Wang, 2001; Liu and Sheng, 2003; Zhang et al., 2005), primarily bitegmic, crassinucellate ovules (Bhatnagar, 1965; Wang, 2001; Zhang et al., 2005) and a Polygonum type of embryo sac development (Yoshida and Michikawa, 1973; Yoshida and Nakajima, 1978; Wang, 2001; Zhang et al., 2005; Sheng et al., 2005).

However, Decaisnea displays three embryological characters that are rarely found in Lardizabalaceae, and are thus of substantial systematic implication. First, the antipodals are persistent and relatively large in Decaisnea, while they are ephemeral and smaller in the other Lardizabalaceae (Bhatnagar, 1965; Yoshida and Michikawa, 1973; Yoshida and Nakajima, 1978; Wang, 2001; Sheng et al., 2005; Zhang et al., 2005). Secondly, the endosperm of Decaisnea is free nuclear, while endosperm is ab initio cellular in most Lardizabalaceae (Johri et al., 1992). Thirdly, the pollen grains of Decaisnea are shed when they are three-celled, while those of most Lardizabalaceae are shed at the two-celled stage (Johri et al., 1992).

With these family-level incongruencies in mind, it is important to note that two of the three embryological characters that distinguish Decaisnea from other Lardizabalaceae are regularly observed in Ranunculales, specifically Ranunculaceae. Like Decaisnea, members of Ranunculaceae have persistent antipodals (Williams and Friedman, 2004) and free nuclear endosperm (Johri et al., 1992). While pollen grains in most genera of Ranunculaceae are shed at the two-celled rather than the three-celled stage (Brewbaker, 1967; Johri et al., 1992), Ranunculaceae have a secretory tapetum, simultaneous microsporocyte meiosis, primarily bitegmic, crassinucellate ovules that are numerous in each carpel, and a Polygonum type embryo sac (Jalan, 1963; Johri et al., 1992), like most Lardizabalaceae. We do not conclude that Decaisnea is more closely related to the Ranunculaceae; however, the embryological characteristics of Decaisnea relative to Lardizabalaceae and other Ranunculales may suggest that the systematic position of Decaisnea calls for further evaluation and elevation.

Thus, it would be premature to make formal taxonomic conclusions before stronger evidence is obtained and before evolutionary trends of the gross morphology are finally elucidated; however, we believe our embryological results indicate that Decaisnea is not a simple ‘genus-level fit’ in the Lardizabalaceae. The presence of persistent antipodals, free nuclear endosperm, and three-nucleate pollen upon shedding are characters unique to Decaisnea within Lardizabalaceae. Such normally conservative characters can be used to circumscribe taxa above the generic rank (Tobe, 1989). As mentioned, based on morphological data, Loconte and Estes (1989) suggest that Decaisnea could be treated as a subfamily named Decaisneoideae, and Loconte et al. (1995) circumscribe Decaisnea as a new family within Lardizabalales. Qin (1989, 1997) has treated Decaisnea as a monogeneric tribe Decaisneeae. In light of the present embryological study on Decaisnea insignis, we believe such statements deserve serious reconsideration, particularly those of Qin (1989, 1997). Our suggestion neither directly confirms nor refutes a chloroplast and nuclear DNA sequence-based molecular phylogeny wherein Decaisnea was resolved as a member of Lardizabalaceae (Hoot et al., 1995a, b, 1999). However, we recommend that our new embryological results be incorporated in future phylogenetic studies.

Conclusions

Decaisnea and other genera of Lardizabalaceae characteristically have tetrasporangiate anthers, a secretory tapetum, simultaneous microsporocyte cytokinesis, primarily bitegmic, crassinucellate ovules and a Polygonum-type embryo sac. However, in the family, only Decaisnea has persistent antipodals, nuclear endosperm, and three-celled pollen upon shedding. Based on these embryological results, we suggest that monospecific Decaisnea is in need of taxonomic re-evaluation and circumscription above the genus level.

ACKNOWLEDGEMENTS

We are very grateful to Professor Yi Ren at the Shaanxi Normal University for providing us with material, and we also thank Ms Jie Wen and Dr Qing Cai for technical assistance. We thank Professor Libing Zhang at the Missouri Botanical Garden, Dr Chunying Xue (Kunming institute of Botany), Dr Thomas B. Friedman (Thompson Rivers University) and Dr Tomas Rodriguez-Riano (Universidad de Extremadura, Spain) for their helpful suggestions on the manuscript. This work was supported by a National Sciences and Engineering Research Council of Canada Discovery Grant (grant number 164375 provided to C.R.F.). The work would not have been possible without laboratory supplies and reagents generously supplied by Professor Yi Ren and the Shaanxi Normal University.

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