Experimental Evaluation of Differences in Plastic Phenotypes Between Cardamine Fallax and C. Occulta: Effects of Seasonality on Phenology and Gross Morphology

ISSN 1346-7565 Acta Phytotax. Geobot. 71 (1): 23–32 (2020) doi: 10.18942/apg.201912

Experimental Evaluation of Differences in Plastic Phenotypes between Cardamine fallax and C. occulta: Effects of Seasonality on Phenology and Gross Morphology

1 2, 3 1,* Mie N. Honjo , Karol Marhold and Hiroshi Kudoh

1Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu, Shiga 520-2113, Japan. *[email protected] (author for correspondence); 2Institute of Botany, Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 23 Bratislava, Slovak Republic; 3Department of Botany, Faculty of Science, Charles University, Benátská 2, CZ-128 01 Praha 2, Czech Republic

Phenological responses in species of Cardamine are often accompanied by morphological changes that result in disagreement among researchers about the taxonomic rank and status of a particular taxon. The gross morphology of two closely related eastern Asian species, Cardamine fallax (O. E. Schulz) Nakai and C. occulta Hornem., was compared by growing plants under controlled photoperiod and vernalization conditions. Response to photoperiod and vernalization in C. fallax and C. occulta explained differences in phenology between the two species under natural conditions. The gross morphology was dis- tinctive between the two species grown under the same conditions, but overlapped when C. fallax was grown in the spring photoperiod-temperature regime and C. occulta grown in the autumn environmental regime. Cardamine fallax is distinct from C. occulta in gross morphology when seasonality in flowering time is taken into account. The findings support the distinction between C. fallax and C. occulta. A survey of herbarium specimens revealed the upper cauline leaves of C. fallax to be moderately or densely hairy, while those of C. occulta were glabrous or sparsely hairy.

Key words: Brassicaceae, Cardamine fallax, C. occulta, chilling treatments, diagnostic characters, flowering phenology, gross morphology, long-day plants, photoperiod, vernalization

Cardamine L. (Brassicaceae) with more than species (Kim et al. 2009). Changing these factors 200 species, is one of the largest genera in the not only changes flowering time, but may also family (Lihová & Marhold 2006, Marhold et al. modify gross morphology of some annual species 2018). Differences in gross morphology, such as of Cardamine so that the phenotype is highly de- growth form and leaf shape, is often used to dis- pendent on seasonality (Kudoh et al. 1993, 1995, tinguish between species of Cardamine, especial- 1996). Species of Cardamine form rosettes after ly between closely related taxa that share flower germination. Prior to flowering, the internodes of and fruit morphology (e.g., Marhold 1996, Kučera the upper stem elongate to form the flower stalk et al. 2010, Šlenker et al. 2018). Gross morpholo- while the internodes of the lower stem near the gy is often plastic, making correct interpretation rosette exhibit minimal growth. At anthesis, the of variation important for taxonomic diagnosis plants have both rosette and cauline leaves. Cau- (Kudoh et al. 1993). It has been reported that line leaves are therefore only present during the many species of Brassicaceae, including species reproductive season. The number, shape and dis- of Cardamine, have flowering times dependent section of the cauline leaves are often used as di- on photoperiod and degree of exposure to pro- agnostic characteristics. The rosette and cauline longed cold (vernalization). Long daylight hours leaves are imparipinnate in Brassicaceae. There- and vernalization promote flowering in many fore, gross morphology in Cardamine is charac- 24 Acta Phytotax. Geobot. Vol. 71

Table 1. Population codes, localities and phenology-related habitat environments of Cardamine fallax and C. occulta populations used in this study. Species Population codes Locality Phenology-related habitat environments

C. fallax KM (Kyoto Pref., Montane habitat) Kamakura, Kameoka-shi, alt. ca. Densely covered by kudzu-vine (Pueraria lobata) during 300 m summer

C. fallax Inamura, Kamiichi-machi, Shaded by shrubs during summer; covered with snow TM (Toyama Pref., Montane habitat) Nakaniikawa-gun, alt. ca. 200 m during winter

C. occulta KP (Kyoto Pref., Paddy field) Iwakura, Sakyo-ku, Kyoto-shi, alt. Submerged from May to August; plowed in late autumn ca. 100 m

C. occulta TP (Toyama Pref., Paddy field) Kamijima, Namericawa-shi, alt. Submerged from May to August; covered with snow ca. 15 m during winter

terized by patterns of stem elongation and branch- phylogeny, and Marhold et al. (2016) determined ing and the shape of the leaflets or sections of pin- the correct name to be C. occulta. While C. flex- nate leaves. uosa is tetraploid, C. occulta is octoploid. They Cardamine fallax (O. E. Schulz) Nakai of Ja- have distinct allopolyploid origins with different pan, Korea, and China (Marhold et al. 2007) was combinations of diploid parental species originally described as C. flexuosa With. subsp. (Mandáková et al. 2014, 2019). Lack of agree- fallax O. E. Schulz from Japan, and recognized as ment on the taxonomic rank and status of C. fal- such by Kitamura (1961) and Kimata (1983). It lax, therefore, is attributable to the close similar- was recognized as a distinct species by Kitagawa ity in gross morphology with C. occulta. (1982) and Kudoh et al. (1993) or as a variety of We compared the morphology of two closely C. flexuosa by Ohwi (1972, 1984), Lee (1996), related eastern Asian annual and biennial spe- Cheo et al. (1987). In the Flora of China (Zhou et cies, C. fallax and C. occulta, by growing them al. 2001) and Flora of Japan (Al-Shehbaz et al. under different vernalization and photoperiod re- 2006), it was placed in synonymy under C. parvi- gimes. Our objective was to determine the re- flora L. Based on an evaluation of the original sponse of C. fallax to photoperiod and vernaliza- materials, Marhold et al. (2007) concluded that it tion. The flowering season of C. fallax in May is should be treated as a species distinct from C. approximately one month later than in C. occulta parviflora and C. flexuosa. (Kudoh et al. 1993). We wanted to determine if Cardamine occulta Hornem. was described photoperiod and vernalization in C. fallax ex- based on material that originated from China and plain the differences in phenology between them. was grown in the University of Copenhagen Bo- The second objective was to determine if varia- tanical Garden (Hornemann 1819). The name has tion in gross morphology is distinct or to what been treated as a synonym of C. flexuosa, but it extent it overlaps with C. occulta under different has recently proven to represent a distinct taxon photoperiods and vernalization conditions. We that consists of Asian populations that had been also surveyed herbarium specimens to evaluate considered to be C. flexuosa (Marhold et al. seasonality in gross morphology of the two taxa. 2016). Cardamine flexuosa With. was described Based on our conclusions we provide a key to based on plants in England, and both the Euro- separate C. fallax from C. occulta. pean and Asian populations had been treated as a single species with a pan-Eurasian distribution (Jalas & Suominen 1994, Zhou et al. 2001, Al- Materials and Methods Shehbaz et al. 2006). Lihová et al. (2006) sug- gested that the eastern Asian populations belong Plant materials to a distinct taxon from C. flexuosa based on cp- We chose two populations each of Cardamine DNA (trnL-trnF region) and the nrDNA (ITS) fallax and C. occulta; one from Toyama Prefec- February 2020 Honjo & al. — Plastic Phenotypes of Cardamine fallax and C. occulta 25 ture and the other from Kyoto Prefecture (Ta- ble 1, Fig. 1). These are the same populations used by Kudoh et al. (1993) to describe phenotypic variation under field conditions. Seeds were collected from 12 plants for each population, from Cardamine occulta in April and from and C. fallax in May. The populations were located in different habitats and climatic zones, the Toyama montane population of C. fallax (TM) and the Toyama paddy field population of C. occulta (TP), on the Japan Sea side of Honshu, which is covered by deep snow in winter. The snow pro- vides milder winter temperatures, thermal insu- lation and dark moist conditions on the ground surface. In contrast, the Kyoto montane population of C. fallax (KM) and the Kyoto paddy field population of C. occulta (KP) are on the Pacific side of Honshu and exposed to dry, freezing conditions during the winter.

Experimental design Parts of the results of the experiments were Fig. 1. Four sampling sites in this study. Toyama montane published previously for the TP and KP popula- (TM) and Kyoto montane (KM) populations of Carda- tions of C. occulta (as C. flexuosa in Kudoh et al. mine fallax and Toyama paddy field (TP) and Kyoto pad- 1995). In the current study, we reanalyzed data dy field (KP) populations of C. occulta. for C. occulta and added the results of unpub- lished data on C. fallax. Growth experiments were conducted under the same conditions simul- °C/15 °C (day/night). Non-chilled plants were taneously for the two species. Seeds were stored transferred to growth cabinets one week after be- at room temperature until they were used in the ing transplanted into pots. We determined the growth experiments. In late August, the seeds photosynthetically active radiation (PAR) in the were sown and germinated in the greenhouse. growth cabinets, ca. 230 ~mol m-2 s-1 on average Two weeks after sowing, eight to ten seedlings at the surface of the pots, by use of a data logger from each parental plant (12 plants /population) (LI-1000, Li-Cor; USA) with LI-190SA sensor were transplanted into plastic pots and cultivated (Li-Cor). Light was supplied by a combination of under four conditions; chilling + long day (C-L), 22, 80-W fluorescent lamps (Toshiba), 9, 400-W non-chilling + long day (NC-L), chilling + short extra high pressure mercury lamps (metal-halide, day (C-S), non-chilling + short day (NC-S). We Yoko, Toshiba) and 12, 200-W reflector lamps grew half of the plants at 4 °C, 12 h day length for (Toshiba). Further details of the experimental 30 days, which mimicked the long term exposure procedures are in Kudoh et al. (1995). to low temperatures during winter. No visible changes (or damage) were observed in the plants Traits examined and data analysis after being subjected to the chilling treatments. We recorded the number of plants in flower Chilled and non-chilled plants were divided into 120 days after transplanting them to the photope- two groups and grown in long-day (16 h day riod chamber. Four phenological traits (days to length) or short-day (8 h day length) growth cabi- bolting; days to flowering; number of leaves on nets (Koito, Model-KG). Air temperature was 20 the main stem; and days to flowering cessation on 26 Acta Phytotax. Geobot. Vol. 71

Fig. 2. Phenological traits of Cardamine fallax and C. occulta under four growth conditions [chilling + long day (C-L), non- chilling + long day (NC-L), chilling + short day (C-S), and non-chilling + short day (NC-S)]. A, Number of plants flowering within 120-day experimental period. Denominators are number of plants examined. B, Days to bolting; C, days to flowering; D, number of leaves on main stem; and E, days to flowering cessation on main inflorescence. Standard deviations indicated by error bars in B–E. the main inflorescence) and eight morphological basal primary branches; number of upper prima- traits [plant height; number of rosette leaves ry branches; and number of leaves on the primary (main stem); number of cauline leaves (main branches] were measured for all plants that flow- stem); maximum internode length; number of ered within the 120-day period. All data analyses February 2020 Honjo & al. — Plastic Phenotypes of Cardamine fallax and C. occulta 27

Fig. 3. Eight gross morphological traits of Cardamine fallax and C. occulta under four growth conditions [chilling + long day (C-L), non-chilling + long day (NC-L), chilling + short day (C-S), and non-chilling + short day (NC-S)]. A, Plant height; B, number of rosette leaves on main stem; C, number of cauline leaves on main stem; D, maximum internode length; E, number of basal primary branches; F, number of upper primary branches; G, number leaves on primary branches; and H, number of leaves on secondary branches. I, Result of Principal component analysis (PCA) for gross morphological traits of C. fallax and C. occulta under four growth conditions. J, Relationship between PC1 score and days to flowering. Stan- dard deviations indicated by error bars in A–H.

and principal component analyses (PCA) were done using R software (v.3.6.1). Results

Morphological survey of herbarium specimens All plants of Cardamine fallax flowered under Based on the results from the growth experi- C-L, NC-L, and C-S, but none flowered under ments, we studied the morphology of herbarium NC-S until the end of the experiment (Fig. 2A). specimens of Cardamine fallax and C. occulta in All plants of C. occulta flowered under all treat- the Kyoto University Museum, Japan (KYO). ments (Fig. 2A). In all four phenological traits 28 Acta Phytotax. Geobot. Vol. 71

Fig. 4. Growth forms of Cardamine fallax and C. occulta under four growth conditions [chilling + long day (C-L), non-chilling + long day (NC-L), chilling + short day (C-S), and non-chilling + short day (NC-S)]. A. TM and KM plants of C. fallax. B. TP and KP plants of C. occulta. Scale bars represent 30 cm. February 2020 Honjo & al. — Plastic Phenotypes of Cardamine fallax and C. occulta 29

Fig. 5. Shapes of leaves of Cardamine fallax and C. occulta in growth experiments with controlled photoperiod and chilling treatments. Two rosette, two middle cauline, and two upper cauline leaves on main stem at flowering stage arranged from left to right in each panel. A. Leaf shapes of TM and KM plants of C. fallax and TP and KP plants of C. occulta grown under chilling + long day (C-L), spring photoperiod-temperature regime. B. Leaf shapes of KP plants growth under four conditions, [i.e., C-L, non-chilling + long day (NC-L), chilling + short day (C-S), non-chilling + short day (NC-S)]. Scale bars represent 5 cm. 30 Acta Phytotax. Geobot. Vol. 71

[i.e., days to bolting, days to flowering, number of gime, but similar when C. fallax in C-L was com- leaves on the main stem, and days to flowering pared with KP of C. occulta under NC-S, the au- cessation on the main inflorescence (Fig. 2B–E)], tumn environmental regime (Fig. 4A & B). Leaf differences between species were much greater shape also showed a similar tendency (Fig. 5); than between populations within a species. In all distinct between species under C-L (Fig. 5A). The four phenological traits, Cardamine fallax gener- shape of the leaves of the KP plants of C. occulta ally lagged C. occulta by two to three times (Fig. under NC-S were similar to the leaf shape of C. 2B–E). The degree of promotion of phenological fallax under C-L (Fig. 5). events was mostly C-L > NC-L > C-S > NC-S We examined 118 specimens of C. fallax and (Fig. 2B–E). 281 of C. occulta deposited in KYO. All the spec- Differences between species were obvious in imens of C. fallax with flowers and/or fruits were some gross morphological traits, but some traits collected in the spring; Mar. (1), Apr. (49), May were highly dependent on the treatment (Fig. 3A– (64), and Jun. (4), while specimens of C. occulta H). The number of rosettes and cauline leaves with flowers and/or fruits were collected all year (Fig. 3B & C) and maximum internode length around with a large peak in spring: Jan. (5), Feb. and number of leaves on the secondary branches (10), Mar. (66), Apr. (112), May (53), Jun. (8), Jul. was distinctively greater in C. occulta (Fig. 3D & (2,) Aug. (5), Sep. (1), Oct. (8), Nov. (8), and Dec. H). In height (Fig. 3A), number of basal branches (3). Cardamine fallax was typically densely hairy (Fig. 3E), and number of leaves on primary throughout, while C. occulta was sparsely hairy branches (Fig. 3G), the distinction between spe- on the stem and leaves, or sometimes the upper cies varied depending on the treatment. The num- stem and leaves were glabrous. The differences ber of upper branches showed no clear differenc- were most conspicuous in the density of hairs on es between species (Fig. 3F). the surface of the upper cauline leaves. The upper The first two principal components (PC) in cauline leaves of C. fallax were moderately to the principal component analysis, PC1 and PC2, densely hairy, while those of C. occulta were gla- explained 62.7% of the total variation (Fig. 3I). brous or sparsely hairy. By combining two PCs, C. fallax and C. occulta were clearly distinguished except for KP under C-S and NC-S (Fig. 3I). The analyses indicated that (NC-S) the two species are morphologically Discussion distinct under C-L, which represents the spring temperature-photoperiod regime (Fig. 3I). How- The experiments clearly showed that Carda- ever, in the autumn regime (NC-S), the overall mine fallax required either long days or exposure gross morphology of C. occulta was similar to to prolonged cold to flower. Both conditions that of C. fallax (Fig. 3I). It appears that gross strongly promote flowering in C. fallax. The lack morphology is partly dependent on phenological of these conditions resulted in continual vegeta- timing, and PC1 is significantly correlated with tive growth for over four months in C. fallax, days to flowering (Pearson’s correlation coeffi- which corresponds with previous reports that cient r = 0.90, P < 0.001, Fig. 3J). Because KP flowering in C. fallax was restricted to the spring plants under C-S and NC-S delayed flowering to while no flowering was observed in autumn un- the level of C. fallax under C-L and NC-L, re- der natural conditions (Kudoh et al. 1993). Even spectively (Fig. 2C), the morphological similarity under C-L conditions, C. fallax flowered after is likely attributable to the similarity in days to producing on average 20.8 and 27.3 leaves, for flowering (Fig. 3J). TM and KM, respectively, which was greater The growth form was distinct between C. fal- than in C. occulta, i.e., 6.1 and 7.4 leaves, in TP lax and C. occulta when they were compared un- and KP, respectively. This explains earlier flow- der C-L, i.e., spring photoperiod-temperature re- ering of C. occulta in the spring under natural February 2020 Honjo & al. — Plastic Phenotypes of Cardamine fallax and C. occulta 31 conditions (Kudoh et al. 1993). In contrast to the occulta reaches a flowering stage earlier than C. situation with C. fallax, C. occulta can bloom in fallax, but autumn-flowering C. occulta delays response to the autumn photoperiod without ex- flowering under short-day conditions. Therefore, periencing cold; corresponding to our observa- if C. occulta is delayed in flowering, it is likely tions of spring and autumn flowering plants ofC. that its morphology becomes similar to C. fallax. occulta under natural conditions. Among the traits we examined, C. fallax is sepa- Analyses of eight traits clearly showed that rated from autumn-flowering C. occulta by hav- gross morphology can reliably be used to distin- ing more upper cauline branches, at least in our guish Cardamine fallax from spring-flowering experiments. In herbarium specimens, the hairs (C-L) C. occulta. Cardamine fallax is distin- on the upper surface of the cauline leaves are di- guished from C. occulta by its greater height, agnostic for separating C. fallax and C. occulta. more rosette leaves, longer internodes, fewer bas- The morphological differences between C. fallax al branches, more leaves on the primary branch- and C. parviflora, and between C. fallax and C. es, and lack of secondary branches. However, we impatiens has been described previously (Mar- observed overlap in gross morphology between hold et al. 2007). C. fallax and autumn flowering plants of C. oc- Our analyses clearly showed that C. fallax is culta. Perhaps this is the reason some taxono- distinct from C. occulta in gross morphology mists treat C. fallax as an infraspecific taxon of when seasonality in flowering time is taken into C. flexuosa, which was considered to include C. account. We believe this supports the treatment occulta at that time (Ohwi 1972, 1984, Kimata of C. fallax as a species separate from C. occulta. 1983, Lee 1996, Cheo et al. 1987). The morpho- Cardamine fallax flowers only in the spring and logical similarity between C. fallax and autumn is morphologically distinct from typical C. occul- C. occulta has also been pointed out by Yonekura ta that flowers at the same time. Although C. fal- (2017). The dependency of PC1 on days to flower- lax and C. occulta with delayed flowering are ing suggests that similarity in gross morphology similar in gross morphology, C. fallax can be dis- depends on similarity in the timing of flowering tinguished by the density of hairs on the surface after germination. Generally, spring-floweringC. of the upper cauline leaves.

Key to Cardamine fallax and C. occulta

1a. Upper cauline leaves moderately to densely hairy; flowering in spring; leaflets of lower and middle cauline leaves dissected (sinus depth / lobe length 0.6–1) ...... C. fallax 1b. Upper cauline leaves glabrous or sparsely hairy; flowering in spring and sometimes in autumn, rarely in summer and winter; often at least some leaflets of lower and middle cauline leaves lobed (sinus depth / lobe length 0.2–0.5), sometimes in summer-, autumn-, and winter-flowering plants (not in spring-flowering plants), all leaflets of lower and middle cauline leaves dissected (sinus length /lobe length 0.4–0.8) ...... C. occulta

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Received August 21, 2019; accepted September 3, 2019