http://orcid.org/0000-0002-7502-1848
ABSTRACT
Lamiaceae is the sixth largest angiosperm family and comprises over 7000 species in 236 genera distributed worldwide. China is one of the diversity centers of Lamiaceae; however, limited chromosome counts are available for Chinese Lamiaceae species. In the present study, chromosome numbers of 32 populations representing 21 species and three varieties of nine genera of Lamiaceae from Southwest China are studied, among which chromosome counts of 17 taxa are reported for the first time. Our results show that most of the species studied are diploids, and are consistent with the previously proposed base numbers. Three species, Isodon ternifolius (2n = 48), Nepeta dentata (2n = 32), and one population of Dracocephalum heterophyllum (2n = 24), are shown to be tetraploids. Chromosome numbers for 56 taxa of 11 genera of Lamiaceae from the Hengduan Mountains Region are obtained from published literature and present study, and the frequency of polyploidy is only 12.5%. Our study further support that polyploidy may have played a minor role in the evolutionary diversification of Lamiaceae in the Hengduan Mountains.
Introduction
With 236 genera and about 7000 species, Lamiaceae is the sixth largest family of flowering plants and is distributed all over the world (Harley et al. Citation2004). Many species of the family are economically important and sources for teak wood, essential oil, and medicine. China has a large and diverse flora of Lamiaceae and contains nearly 1000 species in about 100 genera (Chen and Gilbert Citation1994; Li and Hedge Citation1994). It is considered as one of the six regions of high Lamiaceae diversity (Hedge Citation1992). Genera with a substantial number of Chinese species include Chelonopsis Miq., Dracocephalum L., Elsholtzia Willd., Isodon (Schrad. ex Benth.) Spach, Nepeta L., Phlomoides Moench, Salvia L., and Scutellaria L., most of which have a distribution center in Southwest China, especially in the Hengduan Mountains Region (HMR).
Chromosome number and ploidy level are of significant importance in taxonomic and evolutionary studies (Stace Citation1991; Stuessy Citation2009). According to the Index to Plant Chromosome Numbers (IPCN, http://www.tropicos.org/Project/IPCN) (Goldblatt and Johnson Citation1979–2017) and Chromosome Counts Database (CCDB, version 1.45, http://ccdb.tau.ac.il/home/) (Rice et al. Citation2015), chromosome numbers for over 1500 taxa in about 140 genera of Lamiaceae have been reported. However, few studies focused on the Chinese Lamiaceae, and less for species from Southwest China.
In order to contribute to the taxonomy and systematics of Lamiaceae, we have been working on the cytology of Lamiaceae species from China since 2014 (Xiang, Chen et al. Citation2014, Citation2017; Hu et al. Citation2016). One of the main objectives of present study was to report the chromosome numbers of 24 Lamiaceae taxa in nine genera from Southwest China, where most of these genera are highly diversified and widely distributed, and evaluate the systematic significance of chromosome data for some genera. Furthermore, Nie et al. (Citation2005) showed that only 22% of the 552 taxa from the Hengduan Mountains Region with reported chromosome numbers were polyploids, indicating that polyploidy may have played a minor role in the plant evolution and diversification in this area, which is one of the worldwide global biodiversity hotspots (Myers et al. Citation2000). However, in their study, no chromosome count was available for Lamiaceae from the Hengduan Mountains Region, which includes ca. 300 species of 50 genera (Wang Citation1994). After more than a decade, chromosome numbers of some genera of Lamiaceae have been reported from this region. Thus, another main objective of our study was to further test the hypothesis of Nie et al. (Citation2005) on polyploidy based on currently available chromosome data of Lamiaceae.
Materials and methods
Mericarps of 32 populations belonging to 24 taxa (21 species and three varieties) of Lamiaceae were collected from Southwest China (). Voucher specimens were kept at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN). Taxonomic treatment in this study largely followed Li and Hedge (Citation1994) with the exception of genus Phlomoides and Caryopteris Bunge, for which the most recent treatment of Xiang, Dong et al. (Citation2014) and Xiang et al. (Citation2018) were adopted, respectively.
Table 1. Voucher information and chromosomal data of 24 taxa of Lamiaceae examined in this study.
Figure 1. Mitotic metaphase chromosomes of Lamiaceae taxa from Southwest China. (A) Caryopteris minor (Xiang et al. 1397). (B) C. minor (Xiang et al. 1428). (C) Chelonopsis souliei. (D) Dracocephalum bullatum. (E) D. heterophyllum (Xiang et al. 1425). (F) D. heterophyllum (Xiang et al. 1505). (G) D. propinquum. (H) D. tanguticum (Xiang et al. 1412). (I) D. tanguticum (Xiang et al. 1426). (J) D. tanguticum (Xiang et al. 1537). (K) Eriophyton wallichii. (L) Isodon atroruber. (M) I. coetsa var. cavaleriei. (N) I. enanderianus. (O) I. flabelliformis. (P) I. grandifolius var. atuntzeensis. Scale bars = 5 μm.
Figure 2. Mitotic metaphase chromosomes of Lamiaceae species from Southwest China. (A) Isodon irroratus. (B) I. pharicus. (C) I. pseudo-irroratus (Xiang et al. 1601). (D) I. pseudo-irroratus (Xiang et al. 1625). (E) I. pseudo-irroratus (Xiang et al. 1648). (F) I. scrophularioides. (G) I. tenuifolius. (H) I. ternifolius; (I) Melissa axillaris. (J) Nepeta dentata. (K) N. stewartiana (Xiang et al. 1386). (L) N. stewartiana (Xiang et al. 1603). (M) Phlomoides medicinalis (Xiang et al. 1433). (N) P. medicinalis (Xiang et al. 1462). (O) P. younghusbandii. (P) Scutellaria kingiana. Scale bars = 5 μm.
Fresh growing root tips of 5–10 mm long were cut off from germinated mericarps sowed on wet filter paper. They were pretreated in 2 mM 8-hydroxyquinoline for 4 h at 20°C and then fixed in 45% acetic acid for 10 min at about 2°C. Finally they were macerated in the 1:1 mixture of 1 mol l–1 hydrochloric acid and 45% acetic acid for 20–23 s at 60°C, and stained and squashed in 2% aceto-orcein for over 30 min at room temperature in a moist chamber with 45% acetic acid. The slides were faintly heated under an alcohol frame for 1–2 s before observation (Funamoto and Ogawa Citation2004). For the determination of chromosome numbers, at least five individuals of each population were investigated, and 5–10 cells at somatic metaphase for each individual were counted. Photomicrographs were taken using the Olympus Digital Camera (DP72-SET-A-2) system (Tokyo, Japan).
By consulting the online databases IPCN (Goldblatt and Johnson Citation1979–2017) and CCDB (Rice et al. Citation2015), and collating recent cytological studies on Lamiaceae, available data of the chromosome counts for the nine genera in this study as well as the taxa of Lamiaceae from the Hengduan Mountains Region were collected.
Results
The chromosome numbers of 32 populations representing 24 taxa of nine genera of Lamiaceae from Southwest China (; , ) are determined in the present study, among which chromosome numbers of 15 species and two varieties are studied for the first time. Chromosome numbers and polyploidy level for each genus are discussed below.
Chromosome counts for a total of 56 taxa of 11 genera of Lamiaceae from the Hengduan Mountains are collected (). Only seven taxa (12.5%) are shown to be polyploids, of which six are tetraploids, and the remaining one is a hexaploid.
Table 2. Chromosome data of Lamiaceae from the Hengduan Mountains Region.
Discussion
Chromosome numbers of nine genera of Lamiaceae
Caryopteris Bunge
Caryopteris was traditionally placed in Verbenaceae (Briquet Citation1895; Erdtman Citation1945; Chen and Gilbert Citation1994) but later transferred to Lamiaceae (Cantino Citation1992a, Citation1992b; Wagstaff et al. Citation1998; Cantino et al. Citation1999; Harley et al. Citation2004). The genus consists of 16 Asian species, among which 14 species and two varieties can be found in China (Chen and Gilbert Citation1994). However, both morphological and molecular phylogenetic studies showed that Caryopteris s.l. is polyphyletic (Cantino Citation1992a; Abu-Asab et al. Citation1993; Cantino et al. Citation1999; Shi et al. Citation2003; Xiang et al. Citation2018). The recircumscribed Caryopteris s. str. includes seven species, while the remaining species are distributed among five genera: Discretitheca P.D. Cantino, Pseudocaryopteris P.D. Cantino, Rubiteucris Kudô, Schnabelia Hand.-Mazz., and Tripora P.D. Cantino (Cantino et al. Citation1999; Xiang et al. Citation2018).
Currently, chromosome numbers of two species of Caryopteris s. str. have been reported, i.e. C. incana (Thunb.) Miq. (2n = 26, 52) and C. trichosphaera W.W. Sm. (2n = 26) (Popova and Ceschmedjiev Citation1978; Rice et al. Citation2015; Xiang et al. Citation2017). Both the two populations of C. minor (C. Pei & S.L. Chen ex C.Y. Wu) C.L. Xiang included in our study are shown to have 2n = 26 chromosomes (). Thus, it can be inferred that the base chromosome number of Caryopteris s. str. is x = 13, which differs from the base numbers x = 9, 10 in Pseudocaryopteris (Goldblatt and Johnson Citation1979–2017; Rice et al. Citation2015) and x = 14 in Tripora (Xiang et al. Citation2017). No chromosome number is available for Discretitheca, Rubiteucris, and Schnabelia; hence, their base numbers are still unknown.
Chelonopsis Miq.
As currently circumscribed, Chelonopsis comprises 16 species and two varieties which are endemic to East Asia and distributed mainly in China (13 spp.) and Japan (3 spp.) (Xiang et al. Citation2008, Citation2013). Funamoto (Citation2007) studied the chromosome characters of the three endemic herbaceous species in Japan and found that all the three species have 2n = 32 chromosomes. However, chromosome numbers of the remaining species, especially for most shrubby species occurring in China, are still unknown.
Our results show that Chelonopsis souliei (Bonati) Merr., which is a shrub from China and studied here for the first time, also has a chromosome number of 2n = 32 ()). Though we may infer that the base chromosome number of Chelonopsis is x = 16, further study on more species of the genus is still required.
Dracocephalum L.
Dracocephalum has approximately 70 species, most of which are distributed in the alpine and semidry regions of temperate Asia (Harley et al. Citation2004). The genus has a large variation of chromosome number that ranges from 10 to 70 (2n = 10, 12, 14, 20, 24, 28, 36, 70) with possible base numbers of x = 5, 6, 7 (Budantsev Citation1986; Harley et al. Citation2004).
Half of the species of Dracocephalum occur in China (Li and Hedge Citation1994). However, only two species from China have been studied cytologically: D. moldavica L. (2n = 10, 20) and D. heterophyllum Benth. (2n = 24) (Rice et al. Citation2015). Two populations of D. heterophyllum are included in present study but with different chromosome numbers. The Baxoi population from Xizang has 2n = 24 chromosomes ()), which is consistent with previous studies, while the chromosome number of the Lhünzê population is 2n = 12 ()), indicating the species to be diploid and tetraploid. The chromosome numbers of D. bullatum Forrest ex Diels, D. propinquum W.W. Smith, and D. tanguticum Maxim. are reported here for the first time. All the above three species have 2n = 14 chromosomes (, ). Combing our results with previous studies, we find that the base number is largely congruent within D. sect. Sinodracon C.Y. Wu & W.T. Wang (x = 7), D. sect. Calodracon Benth. (x = 7), and D. ser. Peregrina Schischk. (x = 6).
Eriophyton Benth.
Based on the most recent molecular phylogenetic study of subfamily Lamioideae (Bendiksby et al. Citation2011), Eriophyton was shown to be paraphyletic. After incorporating Alajja Ikonn. and three species of Lamium L., Eriophyton s.l. comprises six species extending from Central Asia to Himalaya (Harley et al. Citation2004; Xu et al. Citation2009; Bendiksby et al. Citation2011).
Here we report the chromosome number of E. wallichii Benth., the type of Eriophyton, to be 2n = 16 ()), which is congruent with the result of Huang et al. (Citation1996). Since chromosome numbers of the other five species remain unknown, more studies on the Central Asian species are needed to compare with the Himalayan ones and further confirm the possible base chromosome number x = 8.
Isodon (Schrad. ex Benth.) Spach
About 100 species are now recognized within Isodon, which is distributed mainly in tropical and subtropical Asia, with two endemic species in Africa (Li Citation1988; Li and Hedge Citation1994; Chen, Hu et al. Citation2017; Chen, Xiang et al. Citation2017). China accommodates ca. 80 species of Isodon and almost 60 species can be found in the Hengduan Mountains Region (Li Citation1988; Li and Hedge Citation1994; Zhong et al. Citation2010).
Chromosome numbers of 40 species and 10 varieties of Isodon have been reported, and most of the taxa have a chromosome number of 2n = 24, while only three polyploids have been found (Goldblatt and Johnson Citation1979–2017; Xiang, Chen et al. Citation2014; Xiang et al. Citation2017; Rice et al. Citation2015). A total of 11 taxa are investigated in our study, seven of which are studied for the first time. Except for I. ternifolius (D. Don) Kudô, all taxa have 2n = 24 chromosomes (–P), (A–G)), further confirming the base chromosome number x = 12 (Yamashiro et al. Citation2005; Xiang, Chen et al. Citation2014; Yu et al. Citation2014).
Our study shows that I. ternifolius is a tetraploid (2n = 48) ()). However, both Mehra and Gill (Citation1972) and Gill (Citation1984) found that populations of I. ternifolius from West Himalaya have 2n = 24 chromosomes. Molecular phylogenetic studies of Isodon showed that I. ternifolius forms a single clade within the genus (Zhong et al. Citation2010; Yu et al. Citation2014), indicating its highly isolated phylogenetic placement. More populations of I. ternifolius need to be investigated to further reveal the diversification of Isodon.
Melissa L.
Melissa is a genus of four species distributed across the Eurasian subcontinent, North Africa, and Macaronesia (Harley et al. Citation2004). Three different ploidy levels were found in the type species M. officinalis L.: diploid (2n = 32), triploid (2n = 48), and tetraploid (2n = 64). Its haploid base number was shown to be x = 16 (Kittler et al. Citation2015). Melissa officinalis is grown worldwide and may originate from the Mediterranean Region or West Asia (Hanelt and Institute of Plant Genetics and Crop Plant Research Citation2001). The other three species, M. axillaris (Benth.) Bakh. f., M. flava Benth., and M. yunnanensis C. Y. Wu & Y. C. Huang, are distributed from East to Southeast Asia. Previous studies (Saggoo and Bir Citation1981, Citation1986) showed that both M. axillaris and M. flava have 2n = 34 chromosomes. Our study reveals that the chromosome number of M. axillaris from Southwest China is 2n = 34 ()), which is in accordance with the result of Saggoo and Bir (Citation1981, Citation1986), indicating a base chromosome number of x = 17. The two different base numbers in Melissa correlate well with the biogeography.
Nepeta L.
Nepeta is one of the largest genera of Lamiaceae, with approximately 300 species widely distributed in Eurasian and extending into North Africa (Jamzad et al. Citation2003; Harley et al. Citation2004). It has a highly diverse chromosome number ranging from 12 to 54 (2n = 12, 14, 16, 18, 22, 24, 26, 27, 30, 32, 34, 36, 42, 54). The base chromosome numbers are x = 6, 7, 8, 9, 11, 13, 15, 17, among which x = 9 is the most frequent count (Goldblatt and Johnson Citation1979–2017; Rice et al. Citation2015).
China accommodates 42 species of Nepeta which are mainly distributed in the southwest area. Chromosomes numbers of only three species from China were reported, i.e. N. micrantha Bunge (2n = 18), N. multifida L. (2n = 12), and N. sibirica L. (2n = 12) (Kong et al. Citation2008; Wang et al. Citation2013; Zheng et al. Citation2015; Duan and Ma Citation2017). Here we investigate the chromosome numbers of N. dentata C.Y. Wu & S.J. Hsuan and N. stewartiana Diels for the first time. Nepeta dentata has 2n = 32 chromosomes ()), indicating it to be a tetraploid with a base number of x = 8. Chromosome numbers of the two populations of N. stewartiana are 2n = 18 (, L)), which is congruent with most species of Nepeta. Further research work could be advanced to provide additional insights into the relationship within Nepeta species.
Phlomoides Moench
Both molecular phylogenetic and morphological studies showed that the traditionally circumscribed Phlomis L. is non-monophyletic, but supported the split of Phlomis s.l. into two separate genera Phlomoides and Phlomis s. str. (Azizian and Cutler Citation1982; Azizian and Moore Citation1982; Ryding Citation2008; Mathiesen et al. Citation2011; Salmaki et al. Citation2012). The recircumscribed Phlomoides contains ca. 164 species distributed from East Europe, Mediterranean, Central and East Asia to Himalaya (Salmaki et al. Citation2012). All Chinese Phlomis, except for the cultivated P. fruticosa L., were transferred to Phlomoides (Xiang, Dong et al. Citation2014).
Phlomoides is known to have a base chromosome number of x = 11, which is distinct from the base number x = 10 in Phlomis (Fang et al. Citation2007). Consistent with previous studies, the two species of Phlomoides included in present study, P. medicinalis (Diels) Kamelin & Makhm. and P. younghusbandii (Mukerjee) Kamelin & Makhm., also have 2n = 22 chromosomes (–O)).
Scutellaria L.
With approximately 360 species, Scutellaria is a subcosmopolitan genus but poorly represented in moist tropical lowlands (Paton Citation1990a, Citation1990b; Harley et al. Citation2004; Zhao et al. Citation2017). As one of the largest genera within Lamiaceae, Scutellaria also has a complex chromosomal variation as at least 14 different chromosome numbers have been found for the genus (Ranjbar and Mahmoudi Citation2013): 2n = 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 44, 60, 88. The basic chromosome number x = 13 is dominant in East and Southeast Asia, x = 12 is dominant in America, while x = 11 is prevailing in North Africa and Eurasia (Ranjbar and Mahmoudi Citation2013).
Though chromosome numbers of over 100 species of Scutellaria have been studied, only a dozen species were collected from China (Ranjbar and Mahmoudi Citation2013). One species of Scutellaria, S. kingiana Prain, is included in our study. The chromosome number of S. kingiana is 2n = 18 ()), which is rare in the whole genus. Only populations of S. baicalensis Georgi from Korea (Lee Citation1970) and S. intermedia Popov from Tajikistan (Astanova Citation1981) were reported to have 2n = 18 chromosomes. Scutellaria kingiana is restricted to Xizang, China, and grows in gravelly sandy soil of alluvial fans (Li and Hedge Citation1994). It is the only species of S. subg. Anaspis (Reching. f.) Juz. from China and distinct in its upper lip of calyx lacking a scutellum (Wu and Li Citation1977; Li and Hedge Citation1994). Similar character can also be found in several species from Central Asia, e.g. S. ghorana Hedge and S. ariana Hedge.
Frequency of polyploidy in Lamiaceae from the Hengduan Mountains
Chromosome numbers of 56 taxa (ca. 20%) of Lamiaceae from the Hengduan Mountains region are summarized (). The frequency of polyploidy of Lamiaceae (12.5%) in the region is much lower than that of all families (22%) counted by Nie et al. (Citation2005). Our result is also consistent with that of the recent study by Sun et al. (Citation2017), which showed that only 27% of Asteraceae species from the Hengduan Mountains with reported chromosome numbers are polyploids. Many studies (e.g. Hanelt Citation1966; Löve and Löve Citation1967, Citation1975; Morton Citation1993) have revealed that polyploids tend to be more prevalent in the arctic or alpine regions; however, we infer that the distribution of diploids and polyploids varies greatly in different area by further comparing with the frequency of polyploidy of Lamiaceae in the West Africa (80%) (Morton Citation1962) and South India (17%) (Bir and Saggoo Citation1985). This situation may depend on the origin of various taxa and their phylogenetic and geographic relationships (Senn Citation1938; Perry Citation1943).
Though limited chromosome data are available for Lamiaceae from the Hengduan Mountains, most of the taxa sampled are representatives of genera that are taxonomically diversified in this area and characterized by regional radiation, such as Isodon, Phlomoides, and Salvia. For genus Isodon, Yu et al. (Citation2014) revealed that it may have experienced a rapid radiation during late Miocene trigged by one of the major uplifts of the Qinghai-Tibet Plateau and subsequent aridification events, resulting in the greatest diversity of the genus in the Hengduan Mountains Region. Except for its significant morphological diversity, Isodon also has members in a variety of habitats in the Hengduan Mountains, ranging from dry-hot valleys to forests and even alpine meadows. Based on present and previous studies (Xiang, Chen et al. Citation2014; Xiang et al. Citation2017), chromosome numbers of approximately half of the species of Isodon have been reported. However, all the 20 taxa collected from the Hengduan Mountains are shown to be diploids (). A similar phenomenon can also be found in some genera with a large diversity in the Hengduan Mountains (Nie et al. Citation2005), e.g. Aconitum L. (Ranunculaceae) and Pedicularis L. (Orobanchaceae). Thus, our study further support the hypothesis of Nie et al. (Citation2005) that allopatric isolation resulted from geographical and ecological heterogeneity and/or reproductive isolation caused by pollination differentiation of insects, rather than polyploidy, may have largely shaped the diversity and endemisms of Lamiaceae species in the Hengduan Mountains Hotspot.
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References
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