A New Classification of Marginal Resin Ducts Improves Understanding of Hard Pine (Pinaceae) Diversity in Taiwan

Flora 209 (2014) 414–425

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Flora

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A new classiﬁcation of marginal resin ducts improves understanding of

hard pine (Pinaceae) diversity in Taiwan

a,b, a,c d d

Chiou-Rong Sheue *, Hsiu-Chin Chang , Yuen-Po Yang , Ho-Yih Liu ,

a,b,e c

Peter Chesson , Fu-Hsiung Hsu

Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road., Taichung 402, Taiwan

Center of Global Change Biology, National Chung Hsing University, 250, Kuo Kuang Road, Taichung 402, Taiwan

Department of Biological Resources, National Chiayi University, 300 Syuefu Road., Chiayi City 600, Taiwan

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan

Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA

A R T I C L E I N F O A B S T R A C T

Article history: Resin ducts provide important characters for classifying the Pinaceae. Here we study Pinus massoniana

Received 18 November 2013

and P. taiwanensis and show that the generally-used term marginal (=external) resin duct, applied to

Accepted 5 June 2014

ducts in needle leaves, needs to be further differentiated into marginal (strongly attaching to the dermal

Edited by R. Lösch

tissue, and lacking the complete ring structure formed by the sheath cells) and submarginal ducts

Available online 28 June 2014

(adjacent to hypodermal cells, with a complete ring structure formed by the sheath cells). On this basis P.

massoniana and P. taiwanensis, which are nearly indistinguishable based on external morphology, are

Keywords:

clearly differentiated. Their similar morphology has led to a long standing debate on where P. massoniana

Hard pines

occurs in Taiwan. Based on this new classiﬁcation of resin ducts, we examined old herbarium specimens

Pinus massoniana

previously identiﬁed as P. massoniana, surveyed current hard pines in Taiwan, and checked historical

Pinus taiwanensis

Submarginal resin duct documents. Needle leaves of these two taxa were studied and compared with the material of P.

Huoyansan massoniana from mainland China as a reference. Pinus massoniana shows almost exclusively marginal

Junjianyan resin ducts, with extremely rare changes along the length of the duct to submarginal positions. In

contrast, P. taiwanensis shows a mixture of medial, submarginal, septal, and rarely endonal ducts, with

occasional changes of the duct between various types. Re-identiﬁcation of specimens showed that only 5

specimens are possible native P. massoniana collected from northern Taiwan and the others are all P.

taiwanensis. The hard pine of Junjianyan is unexpectedly found to be the only currently known P.

massoniana in Taiwan, which is likely a relic of historical afforestation. The hard pine from Huoyansan and

the Coastal Range, which is widely accepted as P. massoniana, is not this species. In addition to the

taxonomic value of the new deﬁnition applied to these two studied taxa, we expect that this approach can

generally be applied distinguishing the respective characters in the genus Pinus and in other conifers.

Introduction species, Harlow (1931) stated that the relative position of the ducts

in the needle may be used as an aid, but that the number of resin

In conifers, resin ducts provide important characters for ducts in the needles is of no particular importance except for a few

classiﬁcation, especially for distinguishing Pinus L. species species that normally only have two or three ducts. Napp-Zinn

(Boratynska and Bobowicz, 2001; Fu et al., 1999; Knopf et al., (1966) classiﬁed four types of resin ducts of the genus Pinus

2012; Li and Keng, 1994; Law et al., 1978; Richardson, 1998; Wu according to their location in the leaf: (1) ducts in contact with the

and Hu, 1997). Resin ducts have also been used to differentiate hypodermis, viz. marginal (=external); (2) ducts surrounded by

Pinus hybrids (Kormutak et al., 1993). For the identiﬁcation of pine chlorenchyma, viz. medial; (3) ducts in contact with the bundle

sheath (endodermis) in the chlorenchyma, viz. endonal; and (4)

ducts inside the bundle sheath. Biswas and Johri (1997) added

another type of resin duct in the mesophyll, viz. septal, referring to

* Corresponding author at: National Chung Hsing University, Department of Life

the duct connected to both endodermis and hypodermis. In this

Sciences, 250, Kuo Kuang Road, Taichung 402, Taiwan. Tel.: +886 4 22857395; fax:

study, we reexamine the marginal resin duct type and show that it

+886 4 22857395.

E-mail addresses: [email protected], [email protected] (C.-R. Sheue). can be divided usefully into two types, which we then use to solve a

http://dx.doi.org/10.1016/j.ﬂora.2014.06.007

C.-R. Sheue et al. / Flora 209 (2014) 414–425 415

long standing mystery on the taxonomy and distribution of hard Pinus massoniana was ﬁrst described by Lambert (1803). He

pines in Taiwan. stated “the specimen represented in this plate (t. 12) is in the

According to the Flora of Taiwan (Li and Keng, 1994), four Banksian Herbarium, having been brought by Mr. Francis Masson

species of pines are native to Taiwan, P. armandii Franch. var. from the Cape of Good Hope, where it was raised by seeds which

mastersiana Hayata, P. morrisonicola Hayata, P. massoniana Lamb. had been sent from China....I have not been able to obtain any fruit

and P. taiwanensis Hayata. The former two species are soft pines of P. masoniana, nor any further information”. Thus, the plate only

(subgenus Strobus) with one vascular trace inside a vascular showed young shoots, needle leaves and male catkins (Lambert,

bundle, and the latter two species are hard pines (subgenus Pinus) 1803). However, it seems strange that it was given the common

with two vascular traces inside a vascular bundle. Of these native name ‘Indian pine' (Lambert, 1803). Since the type specimen of this

pines, P. taiwanensis has the widest distribution in Taiwan (Li and taxon can no longer be found in the BM herbarium, t. 12 (in

Keng, 1975), ranging from altitudes of 680 m to 3100 m in the Lambert 1803) was selected as an iconotype (Farjon, 2010).

Central Range (Sheue et al., 2003). Since P. hwangshanensis Hsia in Pinus massoniana, with three varieties reported in mainland

China was proved to be a synonym of P. taiwanensis (Fu et al.,1999), China (Fu et al.,1999), is regarded a species widely distributed from

its distribution extends to central and eastern China. southwestern and eastern China and Taiwan. According to Li and

Recently, a third hard pine, P. fragilissima Businský, was Keng (1994) it was mostly found in Taiwan in northern parts and

described from southern Taiwan (Businský, 2003). This new hard eastern coastal hills. The so-called P. massoniana, growing in

pine was distinguished by a sparse crown and long cones with lowland central Taiwan (Huoyansan) and the Coastal Range is in

often ﬂat apophyses, but otherwise has very similar morphology to morphology extremely similar to the other Taiwan’s native hard

P. taiwanensis. Thus, we follow Farjon (2010) in treating P. pine P. taiwanensis. These two hard pines are often identiﬁed by

fragilissima as a variety of P. taiwanensis. their collection localities rather than by recognizable and reliable

Table 1

Results of re-identification using resin duct traits of 34 herbarium specimens previously identified as P. massoniana. Only nine specimens (no. 1–7, 9–10) are confirmed as P.

massoniana with only marginal resin ducts.

No. Location (current place) Herb. code/ Collector, notes on specimen Year Resin duct no Reidentiﬁcation no. (type)

1 Sanjiaoyong (Sanxia, Taipei) TAIF2775 Mori, cultivated 1910 6 (6Mar) P. massoniana

2 Taihoku (Taipei) K Price 341 , open hillside, Siuleny (Sintian) 1912 5 (5Mar) P. massoniana

a f

3 Taihoku (Taipei) K Wilson 10,136 , cultivated and also wild around 1918 5 (5Mar) P. massoniana Herinbi

4 Taihoku (Taipei) K Wilson 10,299 , in all probability wild and 1918 9 (9Mar) P. massoniana indigenous

5 Casan (Taipei) TAIF2782 Sasaki, cultivated 1923 6 (6Mar) P. massoniana

6 Taihoku (Taipei) TAI183485 Suzuki 1935 4 (4Mar) P. massoniana

7 Wantan, Sintian (Taipei) TAI013104 Shimizu 3328 1936 4 (4Mar) P. massoniana

8 Taitung (Coastal Range) TAI013112 Yamamoto & Mori YM161 1937 1 (1Mar)? –

9 Nanfengsan (Kaoshiung) PPI00616 RC Zhang 05, cultivated 1968 7 (7Mar) P. massoniana

10 Taichung Park PPI00611 RC Zhang 51, cultivated 1969 8 (8Mar) P. massoniana

11 Yangmingshan (Taipei) PPI31419 MY Ji [–] 2 (2Med) P. luchuensis

12 Nanris, Taito (Nanliao, Taitung, Coastal Range) TAIF2777 Kawakami & Nakahara, not cultivated 1906 4 (2Med 2Mar) P. taiwanensis

a d

13 N. Horisha (Buli, Nantou) K Price 714 , river bank, Niaibam valley 1912 2 (2Med) P. taiwanensis

f e

14 Casan (Taipei) TAIF2780 S. Sasaki, cultivated 1918 6 (6Med) P. thunbergii

15 Peinanda Xi, Taitung (Coastal Range) TAIF2778 S. Sasaki, not cultivated 1920 3 (2Med 1Mar) P. taiwanensis

16 Casan (Taipei) TAI013102 S. Suzuki 1929 3 (3Med) P. taiwanensis

17 Taitung (Coastal Range) TAI114770 Yamamoto & Mori 1937 6 (4Med 2SM) P. taiwanensis

18 Taipei Botanic Garden TAIF166314 LM Zang 1949 3 (2Med 1SM) P. taiwanensis

19 Anmashan TAI196536 TY Yang 1984 3 (2Med 1SM) P. taiwanensis

20 Dabajianshan TNM G0857 CS Yarng 1986 4 (3Med 1SM) P. taiwanensis

f c

21 National Chung Hsing University, Taichung PPI027272 NC Bai cultivated 1987 3 (3Med) P. luchuensis

22 Huoyansan TAI217224 KC Yang 1988 4 (1Med 2Mar P. taiwanensis

1SM)

23 Guangao, Nantou TNM G065 WB Lu 1989 7 (7Med) P. taiwanensis

24 Xinkangshan, Hualian (Coastal Range) TNM G0854 TY Yang et al. 1989 4 (3Med 1Sep) P. taiwanensis

25 Yangmingsan (Taipei) PPI42420 LG Xu 1990 2 (2Med) P. luchuensis

26 Bilusan (between Nantou and Hualian) TNM G017 HS Lin & CK Lin 1990 5 (4Med 1Sep) P. taiwanensis

27 Dongshan, Yilan TNM G052 JS Deng 1991 2 (2Med) P. luchuensis

28 Dongshan, Yilan TNM G054 JS Deng 1991 2 (2Med) P. luchuensis

29 Weiliaoshan (between Kaoshiung and PPI046993 YD Huang 1992 5 (5Med) P. taiwanensis

Pingdong)

30 Xingangshan, Taitung (Coastal Range) TAIF143887 TC Chen 1994 4 (4Med) P. taiwanensis

31 Dalu forest trail (Hsinchu) TNM G0539 QM Wang 1997 6 (4Med 2SM) P. taiwanensis

32 Huoyansan PPI055794 TC Chen 1998 6 (3Med 3SM) P. taiwanensis

33 Huoyansan TNM G0737 TY Yang et al. 2001 5 (2Med 3SM) P. taiwanensis

34 Huoyansan TNM G0880 Tunghai University 2001 4 (2Med 1Sep P. taiwanensis

1SM)

Abbreviations: Mar, marginal resin duct; Med, medial resin duct; Sep, septal resin duct; SM, submarginal resin duct.

Specimens provided by Kew Gardens.

The result from this specimen was excluded because an intact section was not successfully obtained.

This pine might be P. luchuensis, which usually has (2À) 3 (À4) median resin ducts in each needle leaf. It is native to Japan, but occasionally cultivated in Taiwan.

Although this pine has only 2 medial ducts, it is within in the range of the number of resin ducts per needle of P. taiwanensis (2À) 3–6 (À10) (Sheue et al., 2003). In addition,

a group of sclereids was found in the abaxial side of the vascular tissue which is a characteristic of P. taiwanensis (Sheue et al., 2000), but not in P. luchuensis.

This pine might be P. thunbergii, which usually has 3–9 median resin ducts in each needle leaf. f Cultivated.

416 C.-R. Sheue et al. / Flora 209 (2014) 414–425

diagnostic features. However, without reliable identiﬁcation, the Materials and methods

occurrence and distribution of P. massoniana in Taiwan is

questionable. Needle leaves of spur shoots (=short shoots) of purportedly

The relative position of resin ducts in needles of P. taiwanensis Pinus massoniana from both herbaria and fresh specimens from

(with medial, marginal and septal ducts – Masamune and Suzuki, Taiwan were collected and examined by free hand section (Table 1,

1934; Suzuki et al., 1933; Sheue et al., 2000, 2003) has been Fig. 5). The hard pine found at Junjianyan with marginal resin ducts

suggested as a valuable diagnostic character to distinguish it from in Taipei was included as an additional possible population of P.

other morphologically similar species belonging to the section massoniana during this study. Two well-known native taxa, P.

Pinus, subsection Pinus (Gernandt et al., 2005; Chiang et al., 2006), taiwanensis and P. taiwanensis var. fragilissima, respectively from

including P. massoniana (Fu et al., 1999; Kwei and Lee, 1963; Li, Kunyang (Hehuansan, Nantou) and Wulu (Taitung), were collected

1997; Masamune and Suzuki, 1934; Richardson, 1998; Suzuki et al., and examined for comparison. Material of P. massoniana collected

1933; Wu and Hu, 1997; Zhou, 1999) and P. luchuensis Mayr (Li, from Yandangsan, Wanshihsan and Wuyisan in mainland China

1997; Liu et al.,1994). However, recently individuals purportedly of during 2008; and 2009 provided a reference of needle structure.

P. massoniana were found to have the same anatomical and resin Voucher specimens were deposited in the Herbarium of Taiwan

duct characters in needles as those of P. taiwanensis (Liu 1990; Forestry Research Institute (TAIF) in Taipei.

Sheue et al., 2000, 2003). Segments of the middle parts (c. 2 cm in length) of each of three

In this study, we examine old herbarium specimens identiﬁed fascicles of needle leaves (i.e. totaling 6 needle leaves) of 34

as P. massoniana and survey current hard pines of both P. herbarium specimens previously identiﬁed as P. massoniana were

massoniana and P. taiwanensis from ﬁve localities (Junjianyan, free hand sectioned in multiple places along the mentioned length

Huoyansan, Kunyang, Wulu and the Coastal Range) for resin ducts part of the needles. Prior to sectioning, the material was rinsed

as a diagnostic feature. Among these localities, Huoyansan and the with distilled water for about 10–20 min. For fresh materials, the

Coastal Range are places where P. massoniana has been reported same needle parts of each of 10 fascicles of needle leaves of an

(Zhang and Wang, 1986; Li and Keng, 1994) and widely accepted. individual (10 leaves observed) from a study locality were free

Huoyansan Nature Reserve was established in 1989 by the Council hand sectioned in at least three places, giving at least 30 sections

of Agriculture to conserve the remarkable landscape and P. for each individual. In addition, three needles from each individual

massoniana (http://taiwanpedia.culture.tw/web/content? were sectioned along the entire needle to observe changes in resin

ID=1570). We also intensively trace historical taxonomic docu- ducts characters.

ments relevant to Taiwan’s hard pines for a better understanding of Spurr’s resin sections were used to study developmental

the possible occurrence of P. massoniana in Taiwan. differences between different types of resin duct (Fig. 1) in the

Fig. 1. Distinction between marginal and submarginal resin ducts in Pinus needle leaves. A–B and E. marginal resin duct. C–D and F–G. submarginal resin duct. Abbreviations:

Ep: epithelial cell, Epi: epidermis, Hy: hypodermis, L: lumen, Sh: sheath cell, VS: vascular tissue. (A) A needle cross section treated with clearing method and showing seven

marginal resin ducts (arrows). The inset shows that sheath cells of a resin duct (labeled with stars) do not form a complete ring. (B) Longitudinal view of a marginal duct

showing 2-layered cells (Hy and Ep) between the lumen (arrow) and leaf epidermis (Epi). (C) A needle leaf with 2 submarginal ducts (diamond-head arrows), 2 medial ducts

(open arrows) and 1 septal duct (round-head arrow). (D) Longitudinal section of a submarginal duct showing 3–4 cell layers (Hy, Sh and Ep) between the lumen (diamond-

head arrow) and leaf epidermis (Epi). (E) A marginal resin duct tightly embedded into leaf dermal tissue where it connects to hypodermis, and is devoid of typical sheath cells.

(F–G) Submarginal resin ducts with complete ring structures composed of both of sheath and epithelial cells may connect to a 1–2 cell-layered hypodermis beneath the

epidermis. Scale bars: A and C = 100 mm, B and D = 50 mm.

C.-R. Sheue et al. / Flora 209 (2014) 414–425 417

hard pines of Junjianyan and Huoyansan. Three young needle (parenchyma) surrounding the lumen. A marginal resin duct is

leaves (1–1.5 cm in length) mostly embraced by scale leaves of the extremely close to the leaf surface. Only one cell layer of

spur shoots from each of three individuals from these two localities sclerenchyma separates the leaf epidermal cells and the epithelial

were collected. Each young needle leaf was cut into small pieces cells of the resin duct (Fig. 1A, B and E). Based on cell shape and

(2.0 mm) and ﬁxed in 2.5% glutaraldehyde in 0.1 M sodium afﬁnity of stain on the cell wall, these sclerenchyma cells are more

phosphate buffer (pH 7.3) for 4 h at room temperature. After similar to hypodermal cells of dermal tissue than to the sheath cells

three washings in buffer for 30 min each, the specimens were of a resin duct. The outer sheath cells of a marginal resin duct do

postﬁxed in 1% OsO4 in the same buffer for 4 h. After dehydration not form a complete ring structure, which gives the appearance

through an ethanol series, the material was inﬁltrated for 3 days that the duct is embedded in the dermal tissue (Fig. 1A and E).

and embedded in Spurr's resin (DER = 6.0; Spurr, 1969). The A submarginal resin duct is more removed from the leaf surface

embedded material was then polymerized in an oven at 70 C for (Fig. 1C, D, F, and G). Most important, the outer sheath cells remain

12 h. Semithin sections (1 mm) were cut with an Ultracut E intact and form a ring structure. However, the sheath cells directly

Microtome (Reichert-Jung, Wien, Austria) or a MTX Ultramicro- connected to the hypodermal cells may appear somewhat smaller

tome (RMC, Tucson, Arizona, USA) and stained with 1% toluidine and ﬂattened (Fig. 1F). One to two hypodermal cell layers lie

blue for observation. between the epidermal cells and the sheath cells of a submarginal

In addition, the clearing technique was applied for a better resin duct (Fig. 1F and G).

understanding of the arrangement of resin ducts (especially for the Using these deﬁnitions of marginal and submarginal, marginal

sheath cells of resin ducts). The middle parts of three needle leaves resin ducts were found in the needle leaves of P. massoniana from

of each of three individuals from Junjianyan and Huoyansan were mainland China. For the materials collected from Taiwan, marginal

sampled for both cross and longitudinal sections. These samples resin ducts were only found in the hard pine collected from

were boiled with 95% ethanol for bleaching and cleared with 4% Junjianyan in Taipei. These Junjianyan materials show 7–8(À10)

sodium hydroxide at 40 C for 2–3 days, then stained with 1% exclusively marginal resin ducts in a single needle leaf. Moreover,

Safranin O or toluidine blue. All the sections treated with the above the relative position of this marginal resin duct is stable, based on

mentioned methods were observed with a light microscope the observations of cross sections from different positions of the

(Olympus, BH-2, Tokyo, Japan) ﬁtted with a digital camera. same needle leaf and from a longitudinal view, with only rare

changes to submarginal (Fig. 1B). This simple resin duct composi-

Results tion was also found as characteristic of the P. massoniana

specimens from mainland China.

The distinction between marginal and submarginal resin ducts Needle leaves of the other hard pines collected from Taiwan,

including those from Huoyansan, Kunyang, Wulu and the Coastal

Resin ducts of Pinus are composed of two cell types, the outer Range, always showed mixed resin duct composition, including

sheath cells (sclerenchyma) and the inner epithelial cells submarginal (Fig. 1C), medial – in mesophyll – (Fig. 1C), septal –

Fig. 2. Resin ducts in young needle leaves of Pinus massoniana and P. taiwanensis. Abbreviations: Ep: epithelial cell, Epi: epidermis, Hy: hypodermis, L: lumen, M: mesophyll,

N: nucleolus, NL: needle leaf, Sh: sheath cell, SL: sheath leaf, VS: vascular tissue. A–B. P. massoniana. (A) A pair of young needle leaves inside scale leaves showing 7–8 marginal

resin ducts in each leaf (arrows). (B) A close view of A showing that the epithelial cells of marginal resin ducts directly connected to the epidermis. (C–D) P. taiwanensis. (C) A

young needle leaf with marginal ducts (arrows), submarginal ducts (diamond-head arrow) and medial ducts (open arrow). (D) A close view of C showing a marginal duct on

the adaxial leaf surface (arrow) and a marginal duct (diamond-head arrow). Note that the sheath cells (labeled with stars) of a marginal duct do not form a complete ring

structure adjacent to epidermis. Scale bars: A, C = 1 mm, B, D = 500 mm.

418 C.-R. Sheue et al. / Flora 209 (2014) 414–425

adjacent to both hypodermis and endodermis – (Fig. 1C), rarely (Fig. 2B). Based on cell arrangement and cell size, the cells in the

endonal – connected to endodermis –, and extremely rarely, layer between the epidermis and the epithelial layer of the resin

marginal resin ducts. Such mixed composition including two or duct are more similar to hypodermal cells than to sheath cells.

more types of resin ducts in a single needle leaf agrees with the There are 3–5 resin ducts in young needle leaves collected from

previous report of the P. taiwanensis (Sheue et al., 2003). Moreover, Huoyansan (Fig. 2C and D). Different from the hard pine collected

we found that these submarginal ducts in the needles collected from Junjianyan, the cell walls of the epidermis and hypodermis

from the hard pines of Huoyansan, Kunyang, Wulu and the Coastal are signiﬁcantly thickened at this stage. The resin ducts include

Range are less stable than the marginal resin ducts from the hard marginal, submarginal and medial types in a single needle leaf.

pine needles of mainland China and Junjianyan. That means that a Like the resin ducts observed in mature leaves, the position of

submarginal resin duct of P. taiwanensis may occasionally shift its marginal ducts in needle leaves from Junjianyan are stable, while

relative position along the length of a needle leaf to become medial resin ducts in needle leaves from Huoyansan may shift in position

or marginal. gradually from the leaf apex to the base. These position shifts

include: (1) from endonal to submarginal, (2) from submarginal to

Developmental features of marginal and submarginal resin ducts medial, or (3) from marginal to submarginal. Some resin ducts

remain the same type without position shifts along the entire

The young needle leaves collected from Junjianyan show 7–8 needle leaf.

rudimentary resin ducts, with small lumens, between the

epidermis and the mesophyll tissue (Fig. 2A and B ). These resin Re-identiﬁcation of herbarium specimens of P. massoniana

ducts are small and of uniform size in a single needle leaf. The cell

walls of the outer layer of a resin duct (i.e. sheath cells) and the Based on the results above, we conclude that P. massoniana can

hypodermal cells beneath epidermal cells are not thickened at this be distinguished reliably from P. taiwanensis by its resin duct

stage, but some thickened tissues in vascular tissue are visible composition. Pinus massoniana has exclusively the marginal type of

Fig. 3. Characteristics of resin ducts of needle leaves sampled from herbarium specimens previously identiﬁed as P. massoniana. Abbreviations: Ep: epithelial cell, Epi:

epidermis, Hy: hypodermis, L: lumen, VS: vascular tissue. (A) The needle leaf of Price 341 (K, Sintian, Taipei in 1912) showing marginal resin ducts. (B) The needle leaf of Wilson

10,136 (K, Pinglin, Taipei in 1918) showing marginal resin ducts. The inset shows a marginal resin duct with 6 sheath cells (stars labeled). The epithelial cells (a type of

parenchyma cell) are invisible due to shrinkage during specimen processing. (C) The needle leaf of Shimizu Hideo s.n. (TAI 3328, Sintian, Taipei in 1936) showing marginal resin

ducts. (D) The needle leaf of Kawakami and Nakahara s.n. (TAIF 2777, Nanlaio, Taitung in 1906) showing a combination of submarginal (diamond-head arrow) and medial resin

ducts (open arrows). (E) The needle leaf of Price 714 (K, Puli, Nantou in 1912) showing 2 medial resin ducts. (F) The needle leaf of Sasaki s.n. (TAIF 2778, Peinan River, Taitung in

1920) showing a combination of submarginal (diamond-head arrow) and medial resin ducts (open arrows). All scale bars = 100 mm.

C.-R. Sheue et al. / Flora 209 (2014) 414–425 419

resin duct while P. taiwanensis has a mixed resin duct composition ducts (Table 1 Fig. 3A and C). The other 24 specimens previously

that rarely includes marginal resin ducts. Only nine of the 34 identiﬁed as P. massoniana showed mixed resin duct composition,

examined herbarium specimens show exclusively marginal resin with mostly medial and submarginal ducts (Fig. 3D and F). These

Fig. 4. Characteristics of resin ducts of fresh needle leaves of P. massoniana collected from mainland China (A) and hard pines collected from Taiwan (B–H). Symbols of resin

ducts and abbreviations: arrow, marginal; diamond-head arrow, submarginal; open arrow, medial; round-head arrow, septal; curved arrow, endonal, St, stoma. A. Pinus

massoniana collected from Yedangsan showing 5 marginal resin ducts. (B). A needle leaf collected from Junjianyan showing 9 marginal and 1 submarginal resin ducts. (C–D)

Needle leaves collected from Huoyansan showing consistently a combination of medial, septal and submarginal resin ducts. Rarely, a submarginal duct may shift closer to the

epidermis and appear as a marginal duct. (E–F) Needle leaves collected from the Coastal Range showing a combination of submarginal, median and septal resin ducts. Note

that endonal resin ducts (a curved open arrow head in E) may occasionally occur. (G) A needle leaf collected from Wulu showing a combination of submarginal, median and

septal resin ducts. (H) A needle leaf collected from Kunyang showing a combination of submarginal and median resin ducts. All scale bars = 100 mm.

420 C.-R. Sheue et al. / Flora 209 (2014) 414–425

specimens, therefore, are not P. massoniana. These specimens were specimens variously reported that they occurred on open hillsides,

collected from different parts of Taiwan, including Huoyansan and were planted, or in all probability were wild and indigenous.

Taitung. Based on these results, of the herbarium specimens examined,

Among the nine herbarium specimens that we conﬁrm as P. the ﬁrst and last genuine specimens of P. massoniana were

massoniana, ﬁvespecimens were labeled as cultivated(Chang 05, 51, collected in Taiwan in 1910, and 1969, respectively. However, these

Mori s.n.,1910, Sasaki s.n.,1923, Wilson 10,136). Therefore, only four two specimens were both from cultivated trees. The four

specimens of P. massoniana were possibly collected from the wild. herbarium specimens of P. massoniana possibly collected from

These were from northern Taiwan (the old Taihoku region) (Price the wild in Taiwan are dated 1912 (Price 341) to 1936 (Shimizu

341, Shimizu 3328, Suzuki 13,837, Wilson 10,299). Notes on these 3328), a period of only 25 years.

Fig. 5. Proportions of resin duct types in needle leaves of hard pines collected from ﬁve localities in Taiwan. Needle leaves of the hard pine collected from Junjianyan have

overwhelming marginal resin ducts (99.3%) showing that this species is P. massoniana. The other four localities rendered samples having mixed resin duct composition, with

various proportions of submarginal, medial, septal and endonal ducts in the needle leaves, revealing that the pines at these localities are P. taiwanensis. Note that the samples

from Coastal Range have a relative higher proportion of endonal resin ducts (11%), and the samples from Kunyang (2800 m) only have medial and submarginal ducts in their

needle leaves.

C.-R. Sheue et al. / Flora 209 (2014) 414–425 421

Properties of comparison hard pine populations in Taiwan exclusively marginal ducts in needles leaves. Pinus massoniana and

P. densiﬂora Siebold & Zucc. are the two known species having this

Of the ﬁve localities in Taiwan (Coastal Range, Huoyansan, trait in the Old World. The other taxa in the section Pinus reported

Junjianyan, Kuyang and Wulu) speciﬁcally examined for resin duct with marginal ducts in fact have submarginal ducts.

type as a diagnostic character (Figs. 4 and 5), only one, Junjianyan, In a comprehensive study of Pinus, Shaw (1914) using the

has the marginal resin duct type. The hard pine there is conﬁrmed term "external" instead of "marginal", commented that ‘among

as P. massoniana, as detailed above. the hard pines the external duct is characteristic of the Old

The hard pines collected from the other four localities showed World, there being but two American pines with this character

mixed resin duct composition in their needle leaves, with mostly (resinosa and tropicalis). The internal duct is peculiar to hard

medial and submarginal resin ducts, and some septal resin ducts pines of the New World, its presence in Old World species being

(Fig. 4C–H, Fig. 5). Endonal resin ducts were rarely found (Fig. 4E). extremely rare’ (l.c., p. 6). We re-interpret this statement as

The hard pines collected from lower elevation (Coastal Range, meaning that marginal and submarginal ducts in the needle

Huoyansan and Wulu) have more diverse duct types (medial, leaves are characteristic of the Old World hard pines. The

submarginal, septal and rarely endonal, Fig. 4C–G) than those hypothesis that American pines originated from Asian pines is

collected from high elevation (Kunyang, mainly medial and supported by molecular analysis (Seong and Offner, 2013),

submarginal, Fig. 4H). It is noteworthy that the endonal resin suggesting that marginal and submarginal resin ducts are

duct appears more frequently in the needle leaves of the hard pines relatively primitive duct types among the various duct types

collected from the Coastal Range (11%, Fig. 5). Septal resin ducts in the mesophyll of needle leaves of hard pines.

occur more often in hard pines collected from lowlands, such as the It should be noted that a particular resin duct may change its

Costal Range (300 m) and Huoyansan (250 m), less often from relative position along a needle leaf. Serial sections and clearing of

Wulu (700 m, 4%), and not at all from Kunyang (2800 m). The hard needle leaves in this study demonstrated this possibility for

pine collected from Kunyang has a mixed composition of medial P. taiwanensis. This transition phenomenon reveals a developmen-

and submarginal ducts only (Fig. 4F). Except for the higher tal tendency of gradual shift of the relative position of the resin

proportion of endonal ducts in needles from the Coastal Range, ducts toward the leaf surface from the base to the tip of the leaf.

these results are compatible with a study of elevational variation in This change might be due to the activity of the intercalary

resin duct composition in P. taiwanensis in the Tachia River meristem near the leaf base and the pressure of the expanding

watershed, Taiwan (Sheue et al., 2003). We conclude that these mesophyll during the elongation of the needle. Werker and Fahn

four populations of hard pines do not include P. massoniana, and (1969) reported that resin ducts in P. halepensis needles may also

are all likely P. taiwanensis. change position along the length of a needle. However, such

changes in position are extremely rare in the P. massoniana

Discussion material examined in this study where the ducts are almost always

marginal. The strong contrast in positional stability and develop-

Based on comprehensive observations of resin ducts in mature mental pattern between the marginal ducts of P. massoniana and

and young needle leaves with cross and longitudinal sections the submarginal ducts of P. taiwanensis observed in this study may

along entire needles, we found that the widely used term imply a possible difference in needle morphogenesis.

‘marginal’ (i.e. external) resin duct includes two conditions. Here The development of resin ducts in Pinus needle leaves is related

we suggest that ‘marginal’ resin duct needs to be further divided to the leaf growth pattern. Length growth in needles of Pinus is

into ‘marginal’ and ‘submarginal’ types. This distinction can then apical and subapical in the beginning of needle elongation and

serve as a valuable and reliable identiﬁcation character to later intercalary (Napp-Zinn, 1966). The greater part of the leaf

differentiate P. massoniana and P. taiwanensis, and potentially growth is achieved by the activity of the intercalary meristem

other species. situated at the leaf base. Development and differentiation of ducts

The term ‘submarginal’ resin duct was once used, but not occur basipetally (Werker and Fahn, 1969). It has been further

deﬁned, in descriptions of P. bhutanica (Grierson et al., 1980), P. reported that the duct complex, including sheath and epithelial

fragilissima (Businský, 2003) and P. sylvestris (Catling, 2005). Here cells, develops from the mesophyll tissue in P. nigricans Host

we ﬁrst divide the previous ‘marginal’ type into ‘marginal’ and (Heimerdinger, 1951) and P. halepensis Mill. (Werker and Fahn,

‘submarginal’ types based on (1) their locations in a needle leaf 1969). In this study, the relative position and structure of resin

(extremely close to margin and embedded in the hypodermis ducts were similar between young and mature needle leaves, but

versus close to the margin but adjacent to, not embedded in, the the number of resin ducts in young needle leaves may be a little

hypodermis) and (2) the completeness of the ring structure of the less in both P. taiwanensis and P. massoniana. Because most of these

sheath cells (incomplete with some sheath cells missing toward ducts were well differentiated into sheath cells and epithelial cells

the epidermal surface versus complete with sheath cells forming a at the stage of our observations, we could not identify the

ring structure). This distinction is clear and stable, and can serve as developmental origin of the difference between marginal and

an effective identiﬁcation aid. Thus, we propose this expanded submarginal ducts. For this purpose, younger needle leaves less

classiﬁcation, which we expect may be effectively applied than 5 mm long and still enclosed in scale leaves adjacent to spur

generally in the genus Pinus and other conifers. For example, shoots are needed.

Keteleeria davidiana (Bertrand) Beissn., and Picea morrisonicola The resin duct composition of P. taiwanensis needle leaves,

Hayata (Pinaceae) also have marginal resin ducts in needle leaves based on our collections from four localities in Taiwan (Costal

(re-judged from Hsieh, 1958) and Sciadopitys verticillata Siebold & Range, Huoyansan, Wulu and Kunyang), is conﬁrmed to be a mixed

Zucc. (Sciadopityaceae) in cladodes (re-judged from Dörken and one (Businský, 2003; Liu, 1990; Sheue et al., 2003). In order of

Stützel, 2012; Hsieh, 1958). frequency, it consists of medial, submarginal, septal and endonal

It is a pity that marginal and submarginal resin ducts were ducts. Among these ducts, the two lateral medial ducts (near the

lumped together in most previous books and articles. However, ventral surface) occur consistently in each needle, and so were

some excellent illustrations clearly show the difference between termed ‘basic ducts’ of P. taiwanesis by Sheue et al. (2003). Here we

marginal and submarginal ducts. A quick survey of the marginal further suggest that medial and submarginal ducts are the major

ducts in the section Pinus (Gernandt et al., 2005) from previous duct types in needles of P. taiwanesis, although variation can be

illustrations (Hsieh, 1958; Shaw, 1914), revealed very few taxa with found between populations from sites of different geology and

422 C.-R. Sheue et al. / Flora 209 (2014) 414–425

Table 2

Relevant taxonomic literature on native hard pines in Taiwan chronologically arranged.

Year Author or Title Taxon Distribution Note

source

1803 A.B. A description of the genus Pinus P. massoniana Seeds from China planted in the P. massoniana was ﬁrst described. Because

Lambert Cape of Good Hope in South Africa the type specimen of this taxon in the BM

herbarium can no longer be found, t. 12 (in

Lambert 1803) was selected as an

iconotypea

1896 G.L. Mackay From far Formosa, the island, its Pine Northern Taiwan (Tamsui) “Only a few small pines are found, and they

people and missions are seen on the sides of the mountains”

1896 A. Henry A list of plants of Formosa P. massoniana Only occurs in the mountains of “Fuling (Poria cocos) was collected by

the interior southern savages for sale”

1902 F.B. Forbes An enumeration of all the plants P. massoniana Northern Taiwan (Tamsui) [Oldham 554] might be the ﬁrst specimen

and known from China Proper, Formosa, of P. massoniana from Taiwan. Oldham

W.B. Hainan, the Corea, the Luchu visited Formosa in 1864

Hemsley Archipelago, and the Island of

Hongkong

1908 B. Hayata Flora montana Formosae Pinus sp. 1 (aff. P. In Mt. Morrison Reported two unknown hard pines in

thunbergii) In central mountains Taiwan

Pinus sp. 2 (aff. P.

densiﬂora)

1910 T. A list of plants of Formosa P. massoniana – Scientiﬁc name and Japanese name

Kawakami provided

1911 S. Sasaki Plant checklist of Dà tún shan Range P. massoniana Dà tún shan (Taipei) In Japanese

1911 B. Hayata Materials for a Flora of Formosa P. taiwanensis In central mountains, Luantashan P. taiwanensis was ﬁrst described. Type

specimens lodged at TAIF herbarium

1913 B. Hayata Icones plantarum Formosanarum P. brevispica P. brevispica was ﬁrst reported

(vol. 3) P. taiwanensis

1914 S. Nakai Taiwan rinbokushi P. massoniana Tropical and lowland of “This tree has been introduced to southern

P. taiwanensis subtropical, low lands and from northern Taiwan for afforestation and

mountains (translated from protection from wind” (translated from

Japanese) Japanese)

Upper subtropical and temperate

region, in mountains ranging from

central to southern island

(translated from Japanese)

1917 B. Hayata General index to the ﬂora of P. brevispica Xn gao shan, Luán dà shan, Dà wu

Formosa P. massoniana shan, Mái shí shan, Li dòng shan

P. taiwanensis Northern and central Taiwan, Fang

lán shan (Xnhài suìdào), Ma sù

(Jlóng), Qxng tún shan, X kou (Songshan)

Xn gao shan, Shou chéng dà shan,

Néng gao shan

1917 R. Kanehira Formosa trees: indigenous to the P. brevispica The central mountains

island P. massoniana The tree occurs in northern Taiwan

P. taiwanensis (Taipei, Taoyuen), scattered in

groups in secondary forests

The central mountains, near Buli

1923 S. Sasaki The forest plants of hot spring area P. massoniana Caoshan, Beitóu (current Taipei

in Caoshan and Beitóu City)

1925 Y. Shimadai The Plants at Zh shan yán P. massoniana Zhs han yán (current Taipei)

1933 S. Suzuki, Analytical keys to families, genera P. taiwanensis P. brevispica was treated as a synonym of P.

N. Ueyama and species of Formosan tress P. massoniana taiwanensis. P. massoniana has marginal

and M. Hori ducts, P. taiwanensis has external and

medial ducts

1934 G. Vergleichende Anatomie der Blätten P. taiwanensis Resin ducts in needles of P. massoniana are

Masamune der Kiefer in Taiwan P. massoniana marginal, and those of P. taiwanensis are a

and S. mixture of external and medial

Suzuki

1934 Taiwan The completion of the memorial P. massoniana Dà tún shan, Qxng shan Period of afforestation: 1924; 1933

Sanrinkai afforestation in Dàtún Shan Area (also including P.

leuchuensis, P. thungergii)

1936 K. Sawada Professor Otto Warburg, a plant P. sinensis (synonym of P. Táibei, Xndiàn, Yilán low land Warburg visited Taiwan in 1888

collector and researcher in Taiwan massoniana)

1936 R. Kanehira Formosan trees: indigenous to the P. massoniana Northern part (scattered in “Populations of P. massoniana gradually

Islands (revised) (Leaves 10–20 cm long, secondary forests or patches), also decrease from Taipei and Taoyuen to

Fig. 12 showing 5 on Coastal Range in Taitong southern island. Some habitats in Nantou,

marginal resin ducts in a In the Central Ranges (central but none south of Tainan” (translated from

needle) mountains) at 750 m–2800 m Japanese)

P. taiwanensis altitudes, often form pure stands

(Leaves 811 cm long,

Fig. 13 showing 5 ducts in

a needle, including

medial and submarginal

types)

1960 T.J. Liu P. massoniana Northern Taiwan (Ilan, Taipei,

P. taiwanensis Taoyuan) and central (Nantou,

C.-R. Sheue et al. / Flora 209 (2014) 414–425 423

Table2 (Continued)

Year Author or Title Taxon Distribution Note

source

Illustrations of native and Chequangliao, Dajiaxi), and

introduced ligneous plants of eastern mountains (i.e. current

Taiwan Coastal Range)

the Central Ranges 700–3200 m

altitudes, especially in Dajiaxi

1966 T. Liu Study on the phytogeography of the P. massoniana Low land of northern and central

conifers and taxads of Taiwan Taiwan, Coastal Range 300–

1300 m, especially in river banks of

Daanxi and Dajiaxi

1975 H.L. Li and Pinaceae (in Flora of Taiwan, 1st ed.) P. massoniana Mostly in northern parts, also seeds 20 mm long, leaves slender,12–20 cm

H. Keng P. taiwanensis along the eastern Coastal Range long, with 5–9 resin ducts

Frequent in the Central Ranges of seeds 15–18 mm long, leaves more rigid, 8–

750–3000 m, often forming pure 11 cm long, with 4 resin ducts

stands

1986 R. Zhang The ecology of Pinus massoniana in P. massoniana Low land of northern and central Zhang's thesis was ﬁnished in 1979 and

and Z.K. Huoyansan Taiwan, Coastal Range 300– published in 1986

Wang 1300 m

1986 Agriculture ‘Miaoli Huoyansan Nature Reserve’ P. massoniana “The biggest pure stand of P. Based on ‘Cultural Heritage Preservation

of Council, established massoniana in Taiwan in this area” Act’

Executive

Yuan, R. O. C.

1994 H.L. Li and Pinaceae (in Flora of Taiwan, 2nd P. massoniana Mostly in northern parts, also on seeds 20 mm long, leaves slender,12–20 cm

H. Keng ed.) P. taiwanensis eastern Coastal Range long, with 5–9 resin ducts

Frequent in the Central Ranges at seeds 15–18 mm long, leaves more rigid, 8–

altitudes of 750–3000 m, often 11 cm long, with 4 resin ducts

forming large pure stands

2003 R. Businský A new hard pine (Pinus, Pinaceae) P. fragilissima southeastern Taiwan P. fragilissima was ﬁrst described

from Taiwan

2010 Farjon A handbook of the world's conifers P. massoniana Taiwan P. fragilissima was treated as a variety of P.

P. taiwanensis Taiwan taiwanensis because most of the characters

P. taiwanensis var. Taiwan evaluated are either similar to those of P.

fragilissima taiwanensis, or they show over lapping statesa

Farjon, 2010.

This specimen cannot be found in K herbarium.

Here we differentiate this type as submarginal, see results for details.

elevation. However, among this variation, marginal resin ducts listed P. massoniana (no. 1034) as occurring in the mountains of the

remain extremely rare, strongly differentiating P. taiwanesis from P. interior, and claimed that “southern savages” collect the Poly-

massoniana. poraceae fungus Fuling [Wolﬁporia extensa (Peck) Ginns] from this

In summary, two resin duct traits distinguish P. taiwanensis and tree for sale.

P. massoniana: (1) the clear presence of marginal resin ducts in During the Japanese occupation, a second hard pine from

needle leaves of P. massoniana, and their almost complete absence Taiwan was reported, and this was P. taiwanensis (Hayata, 1911).

in needle leaves of P. taiwanensis, and (2) a simple resin duct Hayata (1913) reported a third hard pine (P. brevispica), but this

composition (just one type of resin duct) in the needles of P. species was soon regarded as a synonym of P. taiwanensis (Suzuki

massoniana, and mixed resin duct composition (several different et al., 1933) leaving just two hard pines, P. massoniana and

types of resin duct) in the needles of P. taiwanensis. P. taiwanensis, recognized and widely accepted as native in Taiwan.

In this study we found that most historical specimens Nakai (1914) described P. massoniana as having soft and long

purportedly of P. massoniana were misidentiﬁcations (Table 1). (5–6 in.) needle leaves and occurring in lowlands and mountains of

Only seven of 34 specimens are conﬁrmed as P. massoniana based tropical and lowlands of subtropical regions. According to Nakai

on their resin ducts. Of these, only four were possibly native. All of (1914) P. massoniana was introduced to southern Taiwan from

these were collected from the Taipei area. The other three northern Taiwan for afforestation of forests and as wind breaks.

conﬁrmed specimens of P. massoniana are labeled as cultivated. Our search conﬁrms that no herbarium specimens of

The earliest historical specimens from the wild purportedly P. massoniana exist from non-cultivated sources outside of the

P. massoniana are Price’s two specimens collected in 1912. One Taipei area or later than 1936 (Table 1). Based primarily on

from Taipei is conﬁrmed as P. massoniana, but the other from Puli literature, most western and Japanese botanists reported that

(central Taiwan) is re-identiﬁed as P. taiwanensis. It would be very P. massoniana occurs in the Taipei area or in northern Taiwan

strange that only few specimens, and from a restricted area, were (Table 2). Kanehira (1936) claimed that P. massoniana is distributed

collected if previously P. massoniana had occurred commonly in in northern Taiwan as scattered in secondary forests or patches

Taiwan. and on coastal hills in Taitung (southeastern Taiwan). After the

For a better understanding of the occurrence of P. massoniana, defeat of Japan in 1945, Liu (1960) reported that P. massoniana

Table 2 gives details of the historical documentation related to hard occurs in northern and central Taiwan, and the eastern mountains.

pines in Taiwan. According to Forbes and Hemsley (1902); the ﬁrst Then a different author, Liu (1966) further reported that this tree is

possible specimen of P. massiniana from Taiwan was collected by especially common on river banks of Daanxi and Dajiaxi (central

the last plant collector of Kew Gardens visiting Taiwan, Oldham Taiwan). The Flora of Taiwan (Li and Keng, 1975) stated that

(Oldham 554) around Tamsui (Taipei) in 1864. Unfortunately, this P. massoniana is mostly distributed in northern parts, but also on

specimen can no longer be found in the K herbarium. Henry (1896) eastern coastal hills, and this statement remains in the most

424 C.-R. Sheue et al. / Flora 209 (2014) 414–425

recent edition (Li and Keng, 1994). Zhang and Wang (1986) available diagnostic features, and allow a more certain identiﬁca-

reported on the ecology of purported P. massoniana in Huoyansan tion of taxa.

near Daanxi. Subsequently, the Taiwan government established

the Houyan Mt. Nature Reserve in 1986. Since then, Huoyan Mt. Acknowledgements

(also known as Huoyansan) became widely accepted as having the

largest population of P. massoniana in Taiwan. The authors thank two anonymous reviewers for valuable

Our study of resin ducts implies that the Huoyan Mt. hard pine suggestions improving this manuscript, the K, TAI, TAIF, TNM and

population is P. taiwensis (Table 1, Fig. 5). An isozyme study of the PPI herbaria for providing specimens for study, Messrs Y.L. Huang

hard pine of Huoyansan (Lee, 1990), which was assumed to be P. and K.P. Lo for help collecting materials in mainland China and

massoniana, concluded that this population is genetically distinct Taiwan, respectively, Dr. T.W. Hsu and B. F. Lu for providing

from P. taiwanesis in Taiwan, but this result might be explained by valuable documents and information, Prof. Lee for providing a

the small spatial scale of the Huoyan Mt. population and its relative knife maker and an ultramicrotome, and Miss S.Y. Liao for

isolation from the compared P. taiwanesis populations, which are in translating historical literature in Japanese. This study was

the Central Mountain Range of Taiwan. partially supported by the Ministry of Science and Technology

The only extant population of P. massoniana in Taiwan (MOST-97-2126-B-005-002-MY3) of The Republic of China

conﬁrmed in this study consists of scattered individuals at (Taiwan).

Junjianyan, Taipei. According to Taiwan Sanrinkai (Table 2), large

scale afforestation was conducted around the Taipei area during

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