利用整合分类学方法进行蕨类植物复合体的物种划分: 以线裂铁角蕨复合体为例

doi:10.17520/biods.2019316

摘要/Abstract

摘要：

广泛的杂交和多倍化使得铁角蕨属(Asplenium)下存在着许多分类困难的物种复合体, 针对这些类群进行整合分类学的研究, 有助于我们更加全面和深入地理解物种的界限以及形成机制。线裂铁角蕨复合体(Asplenium coenobiale complex)是铁角蕨属下一个形态多样性较高的类群, 由于缺乏全面取样和系统研究, 该复合体的物种划分长期存在争议。本研究选取线裂铁角蕨复合体中形态变异和地理分布具有代表性的个体, 通过孢粉学研究确定该类群的生殖特性, 运用流式细胞分析获取倍性信息, 同时结合叶绿体和核基因组片段系统发生分析的证据, 对该类群的系统演化关系和起源方式进行了探讨。结果表明: (1)虽然部分孢子囊败育的情况在线裂铁角蕨复合体中十分普遍, 但正常孢子囊内形成的64个孢子说明该类群植物仍能进行正常的有性生殖; (2)该复合体中存在着倍性变异, 其中多角铁角蕨(A. cornutissimum)是二倍体, 而其他成员均为四倍体; (3)依据母系遗传的叶绿体序列所构建的系统发生关系将该类群划为4个分支, 与基于核基因序列构建的系统树存在冲突, 这暗示杂交可能在该复合体的形成过程中起到了重要的推动作用。综上所述, 我们建议将线裂铁角蕨复合体划分为4个物种, 即同源四倍体新种马关铁角蕨(A. maguanense sp. nov.), 二倍体多角铁角蕨, 以及两个由同一对亲本正反交产生的异源四倍体线裂铁角蕨(A. coenobiale)和叶基宽铁角蕨(A. pulcherrimum)。

关键词: 叶绿体基因组序列, pgiC, 线裂铁角蕨复合体, 杂交, 多倍体

Abstract

Due to wide hybridization and polyploidization, there are numerous species complexes with taxonomic challenges in the fern genus Asplenium. Integrative taxonomy using evidence of morphology, cytology and molecular phylogeny provides one of the best ways for the discovery and delimitation of species. The Asplenium coenobiale complex represents one of the spleenwort complexes, which are morphologically variable and difficult in species delimitation. Owing to the lack of comprehensive sampling and systematic study, the taxonomy of this complex remains unresolved. In this study, we selected representative individuals of this complex covering differences in morphology and geography. We conducted a palynological study to explore reproductive characteristics, and inferred the ploidy level through flow cytometry. Furthermore, based on the phylogenetic evidence from chloroplast and nuclear genomes, we discussed the evolutionary relationship and origin of this complex. Our results showed that: (1) The development of 64 spores within a single normal sporangium is indicative of the ability of sexual reproduction, although abortive sporangia are common in the Asplenium coenobiale complex. (2) Ploidy variation is found in this complex, i.e. A. cornutissimum is diploid, whereas other members are all tetraploid. (3) The maternally inherited chloroplast phylogeny supported four clades within this complex, and this was incongruent with the nuclear phylogeny; therefore, it was inferred that hybridization could be an important driving force during the formation of the complex. Based on our analyses, we conduct a revision to the A. coenobiale complex, i.e. one newly discovered autotetraploid species (A. maguanense sp. nov.), one diploid species (A. cornutissimum), and two allotetraploids with reciprocal origins (A. coenobiale and A. pulcherrimum).

Key words: chloroplast sequences, pgiC, Asplenium coenobiale complex, hybridization, polyploidy

梁思琪, 张宪春, 卫然 (2019) 利用整合分类学方法进行蕨类植物复合体的物种划分: 以线裂铁角蕨复合体为例. 生物多样性, 27, 1205-1220. DOI: 10.17520/biods.2019316.

Siqi Liang, Xianchun Zhang, Ran Wei (2019) Integrative taxonomy resolved species delimitation in a fern complex: A case study of the Asplenium coenobiale complex. Biodiversity Science, 27, 1205-1220. DOI: 10.17520/biods.2019316.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2019316

https://www.biodiversity-science.net/CN/Y2019/V27/I11/1205

图/表 9

图1 线裂铁角蕨复合体的叶片分裂程度变异(比例尺均为2 cm)。 (A)叶基宽铁角蕨(8573)全株, 二回羽状至羽状全裂; (B)叶基宽铁角蕨(9525)叶片, 二回羽状至三回羽状-羽状全裂; (C)线裂铁角蕨(9439-1)全株, 二回羽状至羽状半裂; (D)线裂铁角蕨(9524-6)叶, 二回羽状至羽状全裂; (E)线裂铁角蕨(9524-10)全株, 二回羽状至三回羽状-羽状半裂; (F)叶基宽铁角蕨(9443)基部羽片, 三回羽状至四回羽状-羽状全裂; (G)叶基宽铁角蕨(9524-1)基部羽片, 三回羽状至四回羽状-羽状全裂; (H)线裂铁角蕨(9524-17)叶片, 二回羽状至三回羽状-羽状半裂; (I)线裂铁角蕨(9524-4)基部羽片, 三回羽状至羽状全裂; (J)线裂铁角蕨(9524-16)叶片, 二回羽状。

Fig. 1 Variation of the frond division within the Asplenium coenobiale complex (Scale bar = 2 cm). (A) Habit of A. pulcherrimum (8573), bipinnate-pinnatisect; (B) Lamina of A. pulcherrimum (9525), bipinnate to tripinnate-pinnatisect; (C) Habit of A. coenobiale (9439-1), bipinnate-pinnatifid; (D) Frond of A. coenobiale (9524-6), bipinnate-pinnatisect; (E) Habit of A. coenobiale (9524-10), bipinnate to tripinnate-pinnatifid; (F) Basal pinnae A. pulcherrimum (9443), tripinnate to quadripinnatisect; (G) Basal pinnae A. pulcherrimum (9524-4), tripinnate to quadripinnatisect; (H) Lamina of A. coenobiale (9524-17), bipinnate to tripinnate-pinnatifid; (I) Basal pinnae A. coenobiale (9443), tripinnate-pinnatisect; (J) Lamina of A. coenobiale (9524-16), bipinnate.

图2 线裂铁角蕨复合体的采样分布图。DZ、LD、JL、NP、DY、DQK、ML、PA、CL、HL、BZ、GLQ、PJ含义同表1。

Fig. 2 Map of localities of the examined accessions of the Aspelnium coenobiale complex. DZ, LD, JL, NP, DY, DQK, ML, PA, CL, HL, BZ, GLQ, PJ are the same as Table 1.

本研究所采用的分子标记信息

Table 1 Information of locality and sequences of Asplenium involved in this study.All specimens are deposited in PE

表2 本研究所采用的分子标记信息

Table 2 Information of molecular markers in this study

片段 Fragment	引物 Primer	引物序列(5′-3′) Primer sequence (5′-3′)	最佳碱基替代模型 Best substitution model	长度 Length (bp)	参考文献 Reference
trnL-trnF	Fern-1	GGCAGCCCCCARATTCAGGGRAACC	K81UF+G	693	Trewick et al, 2002
	f	ATTTGAACTGGTGACACGAG			Taberlet et al, 1991
rbcL	1F	ATGTCACCACAAACAGA(G/A)ACTAAAGC	GTR+I+G	1,197	Gastony & Rollo, 1995
	1351R	CTTCACAAGCAGCAGCTAGTTCAGGACTCC			Gastony & Rollo, 1995
rpl32-trnP	112F	TCCATCTTAACCGGTCGTCGTTCA	TVM+G	644	Liang et al, 2019
	858R	AGTTTGGTAGCGCGTCATCT			Liang et al, 2019
pgiC	14F	GTGCTTCTGGGTCTTTTGAGTG	HKY+F+G4	648	Ishikawa et al, 2002
	16R	GTTGTCCATTAGTTCCAGGTTCCCC			Ishikawa et al, 2002

图3 线裂铁角蕨复合体的孢子。 (A) (似)线裂铁角蕨(8214), 正常孢子, 孢子外壁具脊(鸡冠状-翅状); (B)(似)线裂铁角蕨(9439-2), 正常孢子, 孢子外壁具脊(鸡冠状-翅状); (C)多角铁角蕨(9444), 正常孢子, 孢子外壁具翅; (D) (似)线裂铁角蕨(8214), 正常孢子(肾形, 饱满)和败育孢子(干瘪); (E) (似)线裂铁角蕨(9439-2), 正常孢子和畸形且发黑的孢子; (F)多角铁角蕨(9444), 正常孢子, 明显小于该复合体中其他类群的孢子; (G)线裂铁角蕨(9524-10), 正常孢子和干瘪、畸形的孢子; (H)线裂铁角蕨(9524-16), 正常孢子和畸形孢子; (I)叶基宽铁角蕨(9524-1), 正常孢子和发黑的孢子; (J)叶基宽铁角蕨(9443), 正常孢子和畸形、发黑的孢子; (K)线裂铁角蕨(8057), 干瘪的败育孢子; (L)线裂铁角蕨(9524-17), 正常孢子, 孢子外壁具脊(鸡冠状-翅状); (M)叶基宽铁角蕨(8059B), 正常孢子, 孢子外壁具脊(鸡冠状-翅状)。A-C, K-M中比例尺为10 μm; D-J中比例尺为50 μm。

Fig. 3 Spores of the Asplenium coenobiale complex. (A) A. aff. coenobiale (8214), a normal spore, perispore lophate (cristate-alate); (B) A. aff. coenobiale (9439-2), a normal spore, perispore lophate (cristate-alate); (C) A. cornutissimum (9444), a normal spore, perispore alate; (D) A. aff. coenobiale (8214), a normal spore (kidney-shaped, well-filled) and abortive spores (shrivelled); (E) A. aff. coenobiale (9439-2), normal spores and several misshapen, blackened spores; (F) A. cornutissimum (9444), normal spores, and the size of which is relatively small compared with other accessions’ spores; (G) A. coenobiale (9524-10), normal spores and several misshapen, shrivelled spores; (H) A. coenobiale (9524-16), normal spores and several misshapen spores; (I) A. pulcherrimum (9524-1), normal spores and several blackened spores; (J) A. pulcherrimum (9443), normal spores and several misshapen, blackened spores; (K) A. coenobiale (8057), shrivelled, abortive spores; (L) A. coenobiale (9524-17), a normal spore, perispore lophate (cristate-alate); (M) A. pulcherrimum (8059B), a normal spore, perispore lophate (cristate–alate). Scale bars in A–Cand K–M are 10 μm; scale bars in D–J are 50 μm.

表3 线裂铁角蕨复合体细胞学和孢粉学研究结果。-: 数据缺失。

Table 3 Results from cytological and palynological examinations of the Asplenium coenobiale complex. -, Data missing.

凭证标本 Voucher specimen	核DNA含量 Nuclear DNA content (pg)		孢子外壁长度 Length of exospore (μm)	推定倍性 Inferred ploidy level	孢子囊内孢子数(统计的孢子囊个数) Spore number per sporangium (Number of checked sporangia)
凭证标本 Voucher specimen	2C	1Cx	孢子外壁长度 Length of exospore (μm)	推定倍性 Inferred ploidy level
A. cornutissimum 9444	11.3 ± 0.5	5.7 ± 0.2	(22-) 25-27-29 (-30)	2x	64 (10)
A. pulcherrimum 9443	15.8 ± 0.1	4.0 ± 0.0	(29-) 32-35-37 (-39)	4x	64 (4)
A. pulcherrimum 9525	16.1 ± 0.5	4.0 ± 0.1	(26-) 30-33-36 (-37)	4x	64 (2)
A. pulcherrimum 8059B	16.7	4.2	-	4x	64 (2)
A. pulcherrimum 9524-1	17.3 ± 0.2	4.3 ± 0.0	(28-) 31-34-38 (-41)	4x	64 (6)
A. coenobiale 9524-13	17.4 ± 0.0	4.3 ± 0.0	-	4x	64 (6)
A. pulcherrimum 9440	17.5 ± 0.2	4.4 ± 0.1	-	4x	-
A. coenobiale 9524-10	17.6 ± 0.4	4.4 ± 0.1	(28-) 29-33-36 (-41)	4x	64 (4)
A. coenobiale 8059A	17.9	4.5	-	4x	-
A. coenobiale 8057	18.3 ± 0.1	4.6 ± 0.0	-	4x	64 (4)
A. coenobiale 9505	18.3 ± 0.1	4.6 ± 0.0	-	4x	64 (3)
A. coenobiale 9524-17	18.3 ± 0.2	4.6 ± 0.0	-	4x	64 (2)
A. coenobiale 9524-16	18.3 ± 0.3	4.6 ± 0.1	(30-) 31-33-35 (-37)	4x	64 (4)
A. coenobiale 9439-1	18.7 ± 0.2	4.7 ± 0.1	-	4x	64 (2)
A. coenobiale 9524-4	18.7 ± 0.4	4.7 ± 0.1	-	4x	64 (4)
A. aff. coenobiale 9439-2	21.2 ± 0.2	5.3 ± 0.1	(28-) 30-33-35 (-39)	4x	64 (1)
A. aff. coenobiale 8214	21.2 ± 0.2	5.3 ± 0.1	(29-) 31-33-35 (-36)	4x	64 (8)
A. pulcherrimum 8245	-	-	(27-) 30-32-35 (-39)	4x	64 (3)
A. pulcherrimum 2016002	-	-	-	-	64 (4)
A. coenobiale 2016017	-	-	-	-	64 (2)

图4 基于叶绿体基因组3个DNA片段进行最大似然分析获得的系统发生树。系统发生分析的支持率表示顺序为: 最大似然法的自展支持率(BSML)和贝叶斯分析的后验概率(PPBI)。图形含义同图2。

Fig. 4 The phylogenetic tree inferred from the maximum likelihood (ML) analysis based on three chloroplast DNA sequences. Numbers above the branches indicate bootstrap values or posterior probability by ML (BSML)/Bayesian inference (PPBI). Meanings of symbols are the same as Fig. 2.

图5 基于核基因pgiC序列进行最大似然分析获得的系统发生树。系统发生分析的支持率表示顺序为: 最大似然法的自展支持率(BSML)和贝叶斯分析的后验概率(PPBI)。图形含义同图2。

Fig. 5 The phylogenetic tree inferred from the maximum likelihood analysis based on pgiC sequence. Numbers above the branches indicate bootstrap values or posterior probability by ML (BSML)/Bayesian inference (PPBI). Meanings of symbols are the same as Fig. 2.

图6 马关铁角蕨(8214)。(A)全株; (B)叶片远轴面; (C)叶片近轴面; (D)叶片基部羽片近轴面; (E)叶片中部羽片远轴面; (F)孢子囊群; (G)末回裂片顶端, 示齿和排水器; (H)鳞片。疑似杂交种(9439-2): (I)生境; (J)全株; (K)末回裂片远轴面。A-C, J-K中比例尺为2 cm; D-H中比例尺为1 mm。

Fig. 6 A. maguanense sp. nov. (8214). (A) Habit; (B) Abaxial side of lamina; (C) Adaxial side of lamina; (D) Adaxial side of basal pinna; (E) Abaxial side of middle pinna; (F) Sorus; (G) Abaxial side of ultimate segment, apex with teeth and hydathodes; (H) Scales. A. aff. coenobiale (9439-2), a suspected hybrid shared half genome with A. maguanense: (I) Habitat; (J) Habit; (K) Abaxial side of ultimate segment. Scale bars in A-C and J-K are 2 cm; scale bars in D-H are 1 mm.

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