Botanical Studies (2010) 51: 257-262.
SYSTEMATICS
Ostryopsis intermedia, a new species of Betulaceae from Yunnan, China
Bin TIAN, Teng-Liang LIU, and Jian-Quan LIU*
Molecular Ecology Group, MOE Key Laboratory of Arid and Grassland Ecology, School of Life Science, Lanzhou University, Lanzhou 730000, Gansu, P.R. China
(Received January 16, 2009; Accepted September 29, 2009)
ABSTRACT. Ostryopsis intermedia B. Tian & J. Q. Liu, a new species of Betulaceae from northwest Yun­nan, China, is described in this study. Morphologically, the new species shows a combination of traits (stable in all examined individuals and populations) of the two recognized species of the genus, which are reproduc-tively isolated from them by differences in flowering phenology and/or geographical distances. In addition, no intraspecific variation was found in the three species' ITS sequences, but there were clear interspecific differ­ences. These results collectively suggest that O. intermedia is sufficiently distinct from the other two described species of Ostryopsis to be recognized as a new species.
Keywords: Ostryopsis; Ostryopsis intermedia; New species.
INTRUDUCTION
Ostryopsis Decne. (Betulaceae) is endemic to China and is represented by two species: O. davidiana Decne., which is widely distributed in northern China; and O. nobilis Bal-four & Smith, restricted to Sichuan and northwestern Yun­nan (Li and Skvortsov, 1999). This genus has been placed together with Carpinus L., Corylus L., and Ostrya Scopoli in the Coryloideae, largely based on the lack of a peri­anth in the staminate flowers (Thorne, 1992; Mabberley, 1997). However, relationships among these genera remain unresolved, particularly concerning the phylogenetic posi­tion of Ostryopsis (Chen et al., 1999; Yoo and Wen, 2007).
In the present study, we report a new species of this endemic genus (Figure 1). During an extensive inves­tigation of most populations of the genus over its en­tire distribution range, we found a few populations in northwest Yunnan previously ascribed to O. nobilis that were morphologically different from the two known species of the genus. In order to evaluate whether these anomalous populations represent a new species, morpho­logical traits, ITS sequences and the flowering phenology of representatives of these populations were analyzed and compared to those of the parapatrically distributed O. nobilis and the geographically more isolated species, O. davidiana. The results collectively suggest that those morphologically anomalous populations are sufficiently distinct to be recognized as a new species.
*Corresponding author: Email: liujq@nwipb.ac.cn. ljqdxy@public.xn.qh.cn; Fax: +86-931-8914288; Tel: +86-971-8914305.
MATERIAL AND METHODS
In the morphological comparisons we examined at least 10 living individuals or herbarium specimens from each of the investigated populations to assess both interspecific morphological differences and the morphological stabil­ity of the putative new species and two known species at the population level. The results are shown in Table 1. In addition, we recorded the flowering and fruiting phenol-ogy of the three species, by monitoring 10 individuals from each of at least three populations of each species at least once per month from March to August in 2007 and 2008. We also outlined the distributional range of each species according to field investigations and specimen records. Woodworth (1930) reported that chromosome of O. davidiana is 2n = 16. In order to count the chromosome numbers of the three species, seeds were collected from their natural populations and germinated in petri dishes lined with moist gauze. The root tips were incubated in colchicine (0.1% w/v) for 2-3 h when they were 0.5 cm long, fixed in Carnoy's fluid (absolute alcohol:glacial ace­tic acid; 3:1) at 4°C for at least 30 min, hydrolyzed in 1 mol/L hydrochloric acid at 60°C for 10 min, then washed with water, stained with carbol fuchsin and squashed for observations. The chromosome numbers were determined from observations of 10 somatic cells.
Three representative trees from each of three distantly distributed populations of each species were randomly chosen for molecular (ITS sequence) identification and comparison (Table 2). Leaves of the selected trees were collected and immediately dried in silica gel until total ge-nomic DNA was isolated from them following the CTAB
258
Botanical Studies, Vol. 51, 2010
method of Doyle and Doyle (1987). The ITS regions of nrDNA were amplified with ITS primers 1 and 4 (White et al., 1990), then the PCR products were purified using a TIANquick Midi Purification Kit following the sup­plier's recommended protocol (TIANGEN). Sequencing reactions were performed with the PCR primers to cover the whole PCR segment using an ABI Prism BigdyeTM Terminator Cycle Sequencing Ready Reaction Kit. The reaction products were analyzed using an Applied Biosys-tems model 3130xl automated sequencer (Perkin Elmer Applied Biosystems). All sequences were submitted to GenBank (accession numbers: GQ250099 to GQ250101), then aligned using ClustalX version 1.81 (Thompson et al., 1997). Carpinus turczaninowii (AF081518) was selected
as an outgroup to root all sequenced individuals. The phy-logenetic relationships of the three species were assessed by maximum likelihood (ML), Neighbor-Joining (NJ) and maximum parsimony (MP) analyses using PAUP* 4.0b10. All gaps (indels) were coded as binary states (0 or 1). ML exhaustive search parameters were: simple addition of se­quences of taxa with TBR branch swapping, MULTREES and COLLAPSE options on. MP analyses (equally weight­ed characters and nucleotide transformations) involved a heuristic search strategy with 100 replicates of random ad­dition of sequences, in combination with ACCTRAN char­acter optimization, MULPARS+TBR branch swapping and STEEPEST DESCENT options on. In these analyses we used bootstrap values (BS) to assess branch support.
Figure 1. Ostryopsis intermedia B. Tian & J. Q. Liu A, fruiting branch; B, fruit; C, seed. Drawn by A. L. Li from B. Tian & J. Q. Liu 2007003 (LZU).
TIAN et al. ― Ostryopsis intermedia, a new species
259
RESULTS
The new species (Ostryopsis intermedia) differs from the two known species of the genus, O. nobilis and O. davidiana, in a number of morphological characters (listed in Table 1). All these characters are stable within each species. The habit and leaf shape of O. intermedia are similar to those of O. nobilis, except that the new species has doubly serrate leaves with acuminate or acute apices (Figure 1), while O. nobilis has irregularly serrate leaves with obtuse or rounded apices (Table 1). Ostry­opsis intermedia flowers and sets fruits early, from April to May, while both O. nobilis and O. davidiana start to flower in June or July (Table 1). Geographically, the new species is found in southwest China and parapatric to O. nobilis, but distantly isolated from O. davidiana (Figure 3). In the only known sympatric site, the new species and O. nobilis showed no overlapping of flowering and fruit­ing periods (Table 1). More than 1000 mature individuals were found at each of the six recorded sites, and more than half of them can normally flower and set seeds. The total distribution range of the new species is similar to that of O. nobilis, and both have limited distributions (Figure 3), although the range of O. davidiana (in northern China) is more extensive. All three species were found to have the same chromosomes number of 2n = 16 (Figure 2).
No intraspecific variation in ITS sequences was found among the nine examined individuals of each species (three from each of three different populations). However, all these sequences are species-specific, with distinct in­terspecific differences, including a total of eight mutations and three indels (Table 3). The new species differs from O. nobilis at nine ITS sites, and from O. davidiana at just three sites (pairwise differences: 1.5% and 0.5%, respec­tively). All phylogenetic analyses produced a similar to­pology with high bootstrap supports for all nodes (100% in all analyses), indicating that the new species is sister to the northerly species, O. davidiana (Figure 4).
DISCUSSION
The results of our molecular investigation suggest that the new species is a sister species to O. davidiana, and O. nobilis is sister to the clade composed of O. davidiana and the new species. Ostryopsis davidiana and O. intermedia share several morphological characters, including acumi­nate or acute leaf apices, doubly serrate leaf margins, and capitate infructescences. However, O. intermedia differs from the northern species in having larger, more densely pubescent leaves that lack resinous glands. The new spe­cies resembles O. nobilis in leaf size, habit and habitat; the main differences between them are in the characters shared
Table 1. Morphological and phenological differences between Ostryopsis intermedia and two known species.
Character
O. davidiana
O. intermedia
O. nobilis
Leaf blade
Ovate or elliptic-ovate, rarely broadly ovate or broadly ob-ovate, margin doubly serrate and usually incised above mid­dle, apex acuminate or acute
Broadly ovate or ovate-orbicu­lar, margin doubly serrate and usually incised above middle, apex acuminate or acute
Broadly ovate or ovate-orbic­ular, rarely ovate, margin irregularly serrate, apex obtuse or rounded
Adaxial leaves
Sparsely white pubescent
Densely white pubescent
Densely yellow tomentose
Leaf size
1.5-5x1-4 cm
3-9 x 2-6 cm
3-10 x 2-6 cm
Leaf resinous gland
Yes
No
No
Infructescence
Capitate
Capitate
Racemose
Fruiting period
July-August
April-May
June-July
Table 2. Locations of populations used for ITS sequence comparison.
Taxon
Location
Lat. (°N)
Long. (°E)
Elevation (m)
Ostryopsis davidiana
Tangchang, GanSu
33°59'
104°28'
1680
Maoxian, SiChuan
31°39'
103°48'
1570
Lingyuan, LiaoNing
40°55'
119°16'
720
O. nobilis
Lijiang YunNan
27°16'
100°13'
1910
Xianggelila, YunNan
27°48'
99°28'
1950
Muli, SiChuan
27°49'
101°12'
2190
O. intermedia
Weixi, YunNan
27°13'
096°04'
1980
Lijiang, YunNan
27°16'
100°13'
1910
Deqin, YunNan
28°22'
98°54'
2870
260
Botanical Studies, Vol. 51, 2010
Figure 2. Mitotic chromosomes of O. intermedia and O. nobilis. A, O. intermedia (2n = 16); B, O. nobilis (2n = 16).
TIAN et al. ― Ostryopsis intermedia, a new species
261
by the new species and O. davidiana (Table 1). Overall, the new species shows a combination of morphological traits (stable in all examined individuals and populations) of the two known species, O. davidiana and O. nobilis, and seems to be an intermediate taxon between them. Hybrids between two differentiated species also generally have intermediate traits between their parental species (Grant, 1981). However, rather than representing a stable and isolated evolutionary lineage (species), hybrid swarms usually show inter-individual morphological variations. In addition, hybrids are usually distributed intermediately between the two assumed parental species and generally have completely or partly overlapping flowering stages with both parents. In contrast, the distributional ranges of the three species studied here suggest that O. davidiana is geographically isolated from both the new species and O. nobilis (Figure 3); the new species is distributed in more southern regions than O. nobilis and although the new spe­cies is parapatric to O. nobilis (one known population oc­curs sympatrically with O. nobilis) neither their flowering nor fruiting periods overlap (Table 1, Figure 3).
Common mechanisms of reproductive isolation include auto- and allo-polyploidy, which usually lead to immedi­ate isolation from ancestral lineages and strongly promote maintenance of the new evolutionary make-up when the new and ancestral lineages have sympatric or parapatric distributions (Grant, 1981). However, this new species cannot have arisen by polyploidy since all three of the species are diploid (Figure 2). Understanding more about the origins and histories of these species is a target of our future research. In addition, since the new species and O. nobilis have limited distributions and wild populations are sparse, both of them warrant high priority for protection for further scientific research.
TAXONOMIC TREATMENT
Ostryopsis intermedia B. Tian & J. Q. Liu, sp. nov.— Type: CHINA. Yunnan: Lijiang, Judian, in thickets on sunny mountain slopes, alt. 1,910 m, 2007-05-09, B. Tian & J. Q. Liu 2007003 (holotype, LZU; isotype, LZU). Figure 1
Species O. nobilis Balfour & Smith. affinis, sed foliis apice acutis, margine duplicato-serratis, infra dense pu-bescentibus albus; infructescentiis capitula differtis. Etiam similis O. davidiana Decne, quae foliis infra sparse pubes-centibus et glandulosis.
Shrubs to 5 m tall. Branches brown or gray, glabrous; branchlets brown, densely gray pubescent, sometimes with red-brown seta. Petiole 2-5 mm, densely pubescent; leaf blade broadly ovate or ovate-orbicular, 3-9 x 2-6 cm, subleathery, abaxially sparsely white pubescent, adaxi-ally densely white pubescent, base cordate or obliquely cordate, margin doubly serrate and usually incised above middle, apex acuminate or acute; lateral veins 6-9 on each side of midvein, raised abaxially. Male inflorescences 1 or 2; bracts densely white pubescent. Female inflorescence
Figure 4. The single most parsimonious phylogenetic tree constructed based on ITS data. Numbers above branches indicate bootstrap supports.
terminal, racemose-capitulate; peduncle 2-3 mm, densely white tomentose; infructescence capitate, bracts forming a tubular sheath, ca. 1-1.5 cm, leathery, densely white pu­bescent, striate, 3-lobed at apex, dehiscent on 1 side when mature. Nutlet brown, ovoid or subglobose, 4-6 x 4-5 mm, shiny, sparsely pubescent, ribbed; Fl. Mar - Apr, fr. Apr-May; Chromosome number: 2n = 16.
Thickets on sunny mountain slopes; 1,500-2,500 m. NW Yunnan.
Key to three species of Ostryopsis
1. Leaf adaxially densely yellow-brown tomentose, margin irregularly serrate, apex obtuse or rounded; infructes-cence racemose................................................O. nobilis
1. Leaf adaxially white pubescent, margin doubly serrate and usually incised above middle, apex acuminate or acute, infructescence capitate.
2. Leaf 1.5-5x1-4 cm, ovate or elliptic-ovate, rarely broadly ovate or broadly obovate, adaxially sparsely white pubescent, with yellow or brown resinous glands......................................................O. davidiana
2. Leaf 3-9 x 2-6 cm, broadly ovate or ovate-orbicular, rarely ovate, adaxially densely white pubescent,
without yellow or brown resinous glands....................
...............................................................O. intermedia
Acknowledgements. This study was supported by grants from the National Natural Science Foundation of China (30725004) and Ministry of Education of China. We thank Dr. Deng Yunfei to write Latin Summary and Miss Li Ai Li to draw figure 1.
LITERATURE CITED
Chen, Z.D., S.R. Manchester, and H.Y. Sun. 1999. Phylogeny
262
Botanical Studies, Vol. 51, 2010
and evolution of the Betulaceae as inferred from DNA sequences, morphology, and paleobotany. Am. J. Bot. 86: 1168-1181.
Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation proce­dure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11-15.
Grant, V. 1981. Plant Speciation. Columbia Univ Press, New York.
Li, P.C. and A.K. Skvortsov. 1999. Betulaceae. In C.Y. Wu and P.H. Raven (eds.), Flora of China, Vol. 2: Science Press, Beijing and Missouri Botanical Garden Press, St. Louis, pp. 284-313.
Mabberley, D.J. 1997. The Plant Book. 2nd ed. Cambridge Uni­versity Press. Cambridge.
Swofford, D.L. 2003. PAUP*: phylogenetic analysis using par­simony (*and other methods), Version 4.0b 10. Sunderland, Massachusetts: Sinauer Associates.
Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins. 1997. The clustal_x windows interface: Flex­ible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic. Acids. Res. 25: 4876-4882.
Thorne, R.F. 1992. Classification and geography of the flowering
plants. Biol. Rev. 58: 226-257.
Yoo, K.O. and J. Wen. 2007. Phylogeny of Carpinus and sub­family Coryloideae (Betulaceae) based on chloroplast and nuclear ribosomal sequence data. Plant Syst. Evol. 267:
25-35.
White, T.J., T. Bruns, S. Lee, and J.W. Taylor. 1990. Ampli­fication and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White (eds.), PCR Protocols: a Guide to Methods and Applications. Academic Press. San Diego, pp.
315-322.
Woodworth, R.H. 1930. Cytological studies in the Betulaceae. 4. Betula, Carpinus, Ostrya, Ostryopsis. Bot. Gaz. 90:
108-115.
中國雲南樺木科一新種：居中虎榛子
田斌 劉騰靚 劉建全
蘭州大學生命科學學院乾旱與草地生態教育部重點實驗室分子生態研究所
本文報導樺木科虎榛子屬一新種：居中虎榛子。該新種形態上位於虎榛子屬已有兩個物種之間，但
這一新的形態特徵組合在所有檢查的個體和居群中都十分穩定。該新種在開花物候以及地理上與已有的
兩個物種存在生殖隔離。ITS 序列在種內不存在變異，種間區別明顯並具種特異性；新種與兩個已知種
之間都存在顯著的 ITS 序列差異。所有的證據都表明應將居中虎榛子獨立為一個新種。
關鍵詞：虎榛子屬；居中虎榛子；新種。