ISSN 1346-7565 Acta Phytotax. Geobot. 71 (1): 1–11 (2020) doi: 10.18942/apg.201913

Recircumscription of Sections of Hemerocallis () from Japan and Adjacent Regions Based on MIG-seq Data

1,† 1,† 1 2 3 Sho Murakami , Koji Takayama , Shizuka Fuse , Shun K. Hirota , Satoshi Koi , 4 5,‡ 1,* Takahito Ideno , Takenori Yamamoto and Minoru N. Tamura

1Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. *[email protected] (author for correspondence); 2Field Science Center, Graduate School of Agricultural Science, Tohoku University, 232-3 Aza-yomogida, Naruko Onsen, Osaki, Miyagi 989-6711, Japan; 3Botanical Gardens, Faculty of Science, Osaka City University, 2000 Kisaichi, Katano, Osaka 576-0004, Japan; 4Botanical Gardens, Graduate School of Science, the University of Tokyo, 3-7-1 Hakusan, Bunkyo-ku, Tokyo 112-0001, Japan; 5Tsushima City Hall, 1441 Kokubu, Izuhara-cho, Tsushima, Nagasaki 817-8510, Japan. ‡Present address: Iriomote Station, Tropical Biosphere Research Center, University of the Ryukyus, 870 Uehara, Taketomi- cho, Yaeyama-gun, Okinawa 907-1541, Japan. †These authors contributed equally to this work

Circumscription of three sections of Hemerocallis that were recognized by Matsuoka & Hotta (Acta Phy- totax. Geobot. 22: 25–43, 1966) and Hotta (Fl. Jap. IVb: 130–134, 2016) was evaluated through MIG-seq analysis of 45 samples of seven species and nine varieties of Hemerocallis, covering all taxa native to Japan except H. fulva vars. aurantiaca and pauciflora. The Neighbor-Net network of the MIG-seq data yielded three major clusters corresponding to the three sections of Hemerocallis recognized by Matsuo- ka & Hotta (1966) and Hotta (2016), except that H. hakuunensis and H. major, which were included in H. sect. Fulvae by Matsuoka & Hotta (1966) and Hotta (2016), were embedded in the cluster of H. sect. Hemerocallis. Hemerocallis hakuunensis and H. major usually have fragrant and lack horizon- tally spreading rhizomes in the same manner as H. sect. Hemerocallis, which are not features of H. sect. Fulvae. We propose a transfer of H. hakuunensis and H. major from H. sect. Fulvae to H. sect. Hemero- callis. Hemerocallis minor appears to be conspecific withH. lilioasphodelus and is better regarded as H. lilioasphodelus var. minor. Hemerocallis lilioasphodelus var. thunbergii is placed in synonym under H. lilioasphodelus var. lilioasphodelus.

Key words: Asphodelaceae, Hemerocallis, H. hakuunensis, H. lilioasphodelus var. minor, H. major, H. sect. Capitatae, H. sect. Fulvae, MIG-seq, Neighbor-Net network,

Hemerocallis L. (Asphodelaceae) comprises vae, Fulvae, Aurantiacae, Capitatae and Anthe- about 13 species and 12 varieties (Hu 1969, Chen latae, based on characteristics of the inflores- & Noguchi 2000, Hotta 2016) of perennial herbs cence, perianth, and color of anthers. Matsuoka & ranging from grasslands near seashores to moun- Hotta (1966) and Hotta (2016) grouped Nakai’s tainous regions in eastern (Webb 1980), ex- sections into three, Hemerocallis, Fulvae, and cept for H. lilioasphodelus L., whose distribution Capitatae, based on color and odor of the flowers extends to Europe. and flowering time and additional morphological Infrageneric classifications of Hemerocallis characteristics. Thus, the infrageneric classifica- were proposed by Bailey (1930), Nakai (1932), tion of the genus Hemerocallis has been quite Matsuoka & Hotta (1966) and Hotta (2016) (Fig. controversial (Fig. 1), but it has never been reex- 1). Bailey (1930) divided the genus into two sec- amined in the light of molecular methods. tions, Euhemera and Dihemera, based on charac- Based on the study of Hotta (2016), six spe- teristics of the inflorescence. Subsequently, Na- cies and 11 varieties are attributed to Japan, al- kai (1932) proposed six sections, Citrinae, Fla- though Kitamura (1964) reported only four spe- 2 Acta Phytotax. Geobot. Vol. 71

Bailey Nakai Matsuoka & This study Species assigned to each section (1930) (1932) Hotta (1966) (2019) recircumscribed in this study and Hotta (2016)

sect. sect. sect. H. citrina, H. hakuunensis Citrinae Hemerocallis Hemerocallis H. lilioasphodelus (=H. minor) sect. H. major sect. Flavae ** Euhemera sect. * sect. sect. Fulvae Fulvae Fulvae H. fulva sect. Aurantiacae sect. sect. sect. sect. Capitatae Capitatae Capitatae H. middendorffii Dihemera sect. Anthelatae

Fig. 1. History of selected infrageneric classifications of Hemerocallis. * Matsuoka & Hotta (1966) considered including Hemerocallis sulphurea in H. aurantiaca. If this were accepted, part of H. sect. Flavae sensu Nakai (1932) would be transferred to H. sect. Fulvae by Matsuoka & Hotta (1966), because Matsuoka & Hotta (1966) included H. aurantiaca (= H. fulva var. aurantiaca) in H. sect. Fulvae. ** Hemerocallis hakuunensis and H. major are transferred from H. sect. Fulvae to H. sect. Hemerocallis in this study. cies and four varieties from Japan. Despite this which has been placed in three sections, were great difference in the treatments between Hotta used in this study (Table 1). Eleven samples were (2016) and Kitamura (1964), the species and their collected in the wild, 24 samples were from the infraspecific taxa have never been evaluated us- Botanical Gardens of Osaka City University and ing molecular methods. 10 samples were from the Koishikawa Botanical The purpose of this study was to use multi- Garden of the University of Tokyo. The plexed inter-simple sequence repeat (ISSR) geno- were dried and stored in silica gel prior to DNA typing by sequencing (MIG-seq) to examine extraction. Identification of the followed Hemerocallis from Japan and adjacent regions to Hotta (2016). Voucher specimens have been de- determine if it would be useful in constructing an posited in the herbarium of Kyoto University infrageneric classification ofHemerocallis and to (KYO). reevaluate the species and varieties. This method Total DNA was extracted from the samples was recently developed by Suyama & Matsuki using the CTAB method (Doyle & Doyle 1987). (2015). Our intent was to use this genome-wide Single-nucleotide polymorphisms (SNPs) were method to elucidate the taxonomic issues based detected by using MIG-seq following the proce- on ample data from single nucleotide polymor- dure by Suyama & Matsuki (2015). Approximate- phisms (SNPs). ly 10 pM of the 2nd PCR product (library) was used for sequencing on an Illumina MiSeq Se- quencer (Illumina), using a MiSeq Reagent Kit Materials and Methods v.3, 150 cycle (Illumina). For data processing, low-quality reads were Leaves of Hemerocallis were collected from removed by the quality_filter option in FASTX- 45 samples from seven species and nine varieties, Toolkit [http://hannonlab.cshl.edu/fastx_toolkit/ February 2020 Murakami & al.—Sections of Hemerocallis Recircumscribed by MIG-seq 3

Table 1. Sources of materials. Sample Species / Variety Locality Source Voucher no. specimen Hemerocallis sect. Hemerocallis 1 Hemerocallis citrina Baroni var. Cult. in Beijing, Bot. Gard., Osaka City Univ. Murakami 28 citrina 2 H. citrina var. vespertina (H. Hara) Japan: Pref. Yamanashi: Takane-cho Koishiwa Bot. Gard., Univ. Tokyo Murakami 50 M. Hotta 3 H. citrina var. vespertina Japan: Pref. Yamanashi: Takane-cho Koishiwa Bot. Gard., Univ. Tokyo Murakami 51 4 H. citrina var. vespertina Japan: Pref. Kyoto: Kyogamisaki Cape, Murakami 52 Kyotango-shi 5 H. citrina var. vespertina Japan: Pref. Wakayama: Rinkai, Shirahama- Bot. Gard., Osaka City Univ. Murakami 36 cho 6 H. citrina var. vespertina Japan: Pref. Nagasaki: Hatakezaki, Tsushima Yamamoto 1007 Isls. 7 H. lilioasphodelus L. var. lilioasphode- Cult. in Cluj-Napoca, Romania Koishiwa Bot. Gard., Univ. Tokyo Murakami 53 lus 8 H. lilioasphodelus var. thunbergii Japan: Hokkaido: Nosappu Cape, Nemuro-shi Bot. Gard., Osaka City Univ. Murakami 25 (Baker) M. Hotta 9 H. lilioasphodelus var. thunbergii Japan: Hokkaido: Shiraoi-cho Koishiwa Bot. Gard., Univ. Tokyo Murakami 54 10 H. minor Mill. China: Mudanjiang Osaka Gakuin Univ. Murakami 45 11 H. minor Cult. in Univ. of Copenhagen Bot. Gard., Bot. Gard., Osaka City Univ. Murakami 55 Denmark 12 H. minor Cult. in Nishinomiya-shi, Pref Hyogo, Japan Bot. Gard., Osaka City Univ. Murakami 10

H. sect. Capitatae Nakai 13 H. middendorffii Trautv. & C. A. Mey. Japan: Hokkaido: Wakasakanai Seashore, Koishiwa Bot. Gard., Univ. Tokyo Murakami 59 var. middendorffii Toyotomi-cho 14 H. middendorffii var. esculenta Japan: Pref. Aomori: Utozaka, Sannohe-machi Koishiwa Bot. Gard., Univ. Tokyo Murakami 56 (Koidz.) Ohwi 15 H. middendorffii var. esculenta Japan: Pref. Toyama: Mt. Yakushi, Toyama-shi Bot. Gard., Kyoto Univ. Tamura et al. 44001 16 H. middendorffii var. esculenta Japan: Pref. Nagano: Shigakogen, Murakami 46 Yamanouchi-machi 17 H. middendorffii var. esculenta Japan: Tokyo: Kyodo-no-Mori, Fuchu-shi Koishiwa Bot. Gard., Univ. Tokyo Murakami 57 18 H. middendorffii var. exaltata (Stout) Japan: Pref. Akita: Otakizawa, Oga-shi Murakami 23 M. Hotta 19 H. middendorffii var. exaltata Japan: Pref. Yamagata: Hoki, Tobishima Isl. Bot. Gard., Osaka City Univ. Murakami 58 20 H. middendorffii var. exaltata Japan: Pref. Yamagata: Arasaki, Tobishima Isl. Bot. Gard., Osaka City Univ. Murakami 16 21 H. middendorffii var. exaltata Japan: Pref. Yamagata: Sai-no-kawara, Bot. Gard., Osaka City Univ. Murakami 17 Tobishima Isl. 22 H. middendorffii var. exaltata Japan: Pref. Yamagata: Sai-no-kawara, Bot. Gard., Osaka City Univ. Murakami 14 Tobishima Isl. 23 H. middendorffii var. exaltata Japan: Pref. Yamagata: Tateiwa, Tobishima Isl. Bot. Gard., Osaka City Univ. Murakami 19 24 H. middendorffii var. exaltata Japan: Pref. Niigata: Koda Seashore, Sado Isl. Murakami 4 25 H. middendorffii var. exaltata Japan: Pref. Niigata: Kasugazaki, Sado Isl. Bot. Gard., Osaka City Univ. Murakami 18 26 H. middendorffii var. exaltata Japan: Pref. Niigata: Senago, Sado Isl. Bot. Gard., Osaka City Univ. Murakami 26

H. sect. Fulvae Nakai 27 H. fulva L. var. fulva Nepal: the Bot. Gard., Osaka City Univ. Murakami 8 28 H. fulva var. fulva China Bot. Gard., Osaka City Univ. Murakami 24 29 H. fulva var. fulva Thailand: Ban Huai Yot, Doi Nang Bot. Gard., Osaka City Univ. Murakami 6 30 H. fulva var. disticha (Donn ex Sweet) Japan: Pref. Saitama: Furutera, Ogawa-machi Koishiwa Bot. Gard., Univ. Tokyo Murakami 60 M. Hotta 31 H. fulva var. disticha Japan: Pref. Osaka: Kawakami, Bot. Gard., Osaka City Univ. Murakami 35 Kawachinagano-shi 32 H. fulva var. disticha Japan: Pref. Hyogo: Nagasaka, Sanda-shi Bot. Gard., Osaka City Univ. Murakami 38 33 H. fulva var. disticha Japan: Pref. Kochi: Togano, Sakawa-cho Bot. Gard., Osaka City Univ. Murakami 31 34 H. fulva var. kwanso Regel Japan: Pref. Kyoto: Kamo River, Kyoto-shi Murakami 33 35 H. fulva var. kwanso Japan: Pref. Kochi: Tengukogen, Tsuno-cho Murakami 3 36 H. fulva var. kwanso Thailand Bot. Gard., Osaka City Univ. Murakami 29 37 H. fulva var. littorea (Makino) Japan: Pref. Kanagawa: Tsurugizaki Cape, Koishiwa Bot. Gard., Univ. Tokyo Murakami 61 M. Hotta Miura-shi 38 H. fulva var. littorea Japan: Pref. Kochi: Ashizuri Cape, Bot. Gard., Osaka City Univ. Murakami 37 Tosashimizu-shi 39 H. fulva var. littorea Japan: Pref. Kagoshima: Kawamukae, Koishiwa Bot. Gard., Univ. Tokyo Murakami 62 Nishinoomote-shi 40 H. fulva var. longituba (Miq.) Maxim. Japan: Pref. Gifu: Hidatakayama Bot. Gard., Osaka City Univ. Murakami 34 41 H. fulva var. sempervirens (Araki) Japan: Pref. Okinawa: Okawa, Nago-shi Murakami 49 M. Hotta 42 H. hakuunensis Nakai Japan: Pref. Nagasaki: Shiratakedaira, Bot. Gard., Osaka City Univ. Murakami 7 Tsushima Isls. 43 H. hakuunensis Japan: Pref. Nagasaki: Izuharamachi-shiine, Yamamoto 424 Tsushima Isls. 44 H. hakuunensis : Gyeongsangnam-do: Mt. Jiri Bot. Gard., Osaka City Univ. Murakami 30 45 H. major (Baker) M. Hotta Japan: Pref. Fukuoka: Mii-hatazaki, Kurume- Bot. Gard., Osaka City Univ. Murakami 5 shi 4 Acta Phytotax. Geobot. Vol. 71 index.html (accessed 8 Dec. 2017)] under the set- major clusters, I, II, and III, and differed only in tings of q = 30 and p = 40. Extremely short se- the resolution of the subclusters. The Neighbor- quence reads were then removed by Tagdust Net network based on 6,769 SNPs data with the (Lassmann et al. 2009) following Suyama & highest resolution among the samples is shown in Matsuki (2015). SNPs were called by Stacks Fig. 2. v. 1.4.4 (Catchen et al. 2013) using all the remain- Cluster I comprised all samples of Hemero- ing reads. For ustacks, the minimum depth option callis middendorffii (sample nos. 13–26). This for creating a stack and the maximum distance al- cluster included one subcluster: [A] H. midden- lowed to align secondary reads to primary stacks dorffii var. esculenta (14–17) and var. exaltata was set to 3 (-m 3) and 1 (-N 1), respectively. The (18–26). In this subcluster, the four samples of gapped assembly option (—gapped: alignments var. esculenta (14–17) and the nine samples of permitting gaps between stacks) was set. The de- var. exaltata (18–26) were loosely grouped. The fault settings were used for other options. For two groups were not mixed together (Fig. 2). cstacks, the number of mismatches allowed be- Hemerocallis middendorffii var. middendorffii tween sample loci when building the catalog was (13) was included in cluster I, but excluded from set to 2. Default settings were used for other op- subcluster [A] (Fig. 2). tions. To select SNPs for further analyses, three Cluster II included all samples of Hemerocal- parameters for population modules in Stacks lis fulva (27–41), and consisted of two subclus- were set as follows: 1) Minimum number of pop- ters: [B] of H. fulva var. disticha (30–33), var. lit- ulations (‘sample’ in this study) at a locus (-p) was torea (38, 39) and var. longituba (40), and [C] of set as 4, 8, 12, 16, and 20. 2) Minimum minor al- H. fulva var. fulva (27–29), var. kwanso (34–36), lele frequency (—min-maf) was set as 0.04. 3) var. sempervirens (41) and one sample of H. fulva Maximum observed heterozygosity (—max-obs- var. littorea from the Miura Peninsula in Kana- het) was set as 0.6. To investigate the effects of gawa Prefecture (37) (Fig. 2). the different parameter settings in the pipeline, Cluster III included samples of five species: datasets resulting from five different thresholds Hemerocallis citrina, H. lilioasphodelus, H. mi- for the minimum number of samples per locus nor, H. hakuunensis, and H. major (1–12, 42–45). were analyzed. The option to restrict data analy- In this cluster, three subclusters were recognized: sis to only the first SNP per locus (—write-sin- [D] H. citrina var. citrina (1) and var. vespertina gle-snp) was used. (2–5), [E] of H. lilioasphodelus var. lilioasphode- We employed the software SplitsTree4 v.4.10 lus (7), var. thunbergii (8, 9) and H. minor (10- (Huson & Bryant 2006) to construct a Neighbor- 12), and [F] of H. hakuunensis (42–44) and one Net network (Bryant & Moulton 2004) for all sample of H. citrina var. vespertina from Tsushi- samples using the uncorrelated P distance matrix ma in Nagasaki Prefecture (6) (Fig. 2). Hemero- calculated from the SNPs matrix. callis major (45) was included in cluster III apart from other subclusters [D]−[F] (Fig. 2). The pairwise genetic distances between sam- Results ples is shown in Appendix 1. The average genetic distances for the subclusters were: A, 0.0954; B, The average number of reads obtained from 0.0998; C, 0.1954; D, 0.1592; E, 0.1387; and F, 45 samples were 139,528. After the filtering steps 0.1457 (Table 2). in Stacks, the number of SNPs detected were 6,769 (minimum number of samples for -p = 4), 3,240 (-p = 8), 1,736 (-p = 12), 864 (-p = 16) and Discussion 442 (-p = 20). The resulting trees of the Neighbor- Net network with different numbers of SNPs Sectional level were almost identical in topology showing three Clusters I, II and III corresponded to the three February 2020 Murakami & al.—Sections of Hemerocallis Recircumscribed by MIG-seq 5

Fig. 2. Neighbor-Net network derived from 45 samples of Hemerocallis, based on uncorrected P distance calculated by 6,769 SNPs. Labels of terminal branches are consistent with sample numbers in Table 1. Blue, orange and green for sample num- bers correspond to sections recognized by Matsuoka & Hotta (1966) and Hotta (2016), i.e. H. sect. Capitata, H. sect. Ful- vae, H. sect. Hemerocallis, respectively. Large circular arcs of I, II, III indicate sections recircumscribed in this study, i.e. H. sect. Capitata, H. sect. Fulvae, H. sect. Hemerocallis. Small circular arcs of [A]–[F] show as follows: [A] H. middendorf- fii var. esculenta and var. exaltata, [B] H. fulva var. disticha, var. littorea and var. longituba, [C] H. fulva var. fulva, var. kwanso and var. sempervirens, [D] H. citrina, [E] H. lilioasphodelus and H. minor, and [F] H. hakuunensis. * This is a sample of H. fulva var. littorea from Miura Pen., Kanagawa Pref., which is separated from subcluster [B] with other sam- ples of H. fulva var. littorea. ** This is a sample of H. citrina var. vespertina from Tsushima Isls., Nagasaki Pref., which was excluded from group [D], which includes other samples of H. citrina. Scale bar represents uncorrected P distance of 0.1.

Table 2. Averages of pairwise genetic distances between samples in each of the subclusters A, B, C, D, E and F, which are circumscribed in Fig. 2. The pairwise genetic distances between samples are indicated in Appendix 1.

Section Subcluster Number of samples Average distance Capitatae A 13 0.0954 Fulvae B 7 0.0998 C 8 0.1954 Hemerocallis D 5 0.1592 E 6 0.1387 F 4 0.1457 6 Acta Phytotax. Geobot. Vol. 71 sections of Hemerocallis (Capitatae, Fulvae and ers. Hemerocallis sect. Fulvae also has horizon- Hemerocallis) recognized by Matsuoka & Hotta tally spreading rhizomes and a tawny V-shaped (1966) and Hotta (2016), with the exception that blotch at the base of the inner tepals, while H. H. hakuunensis and H. major which were includ- sect. Hemerocallis lacks them. This findings also ed in sect. Fulvae by Matsuoka & Hotta (1966) suggest that vegetative organs, such as the rhi- and Hotta (2016) were in cluster III (sect. Hem- zomes are also important for the classification of erocallis). Hemerocallis. Based on the circumscription of Matsuoka & It is interesting that no species share features Hotta (1966) and Hotta (2016), Hemerocallis sect. of two sections of Hemerocallis based on the Fulvae consists of species with pale orangish yel- MIG-seq data, although introgressive hybridiza- low or red, diurnal, often odorless, flowers, while tion between sections Hemerocallis and Fulvae H. sect. Hemerocallis comprises species with have been reported (Nakao & Yamashita 1956, lemon or pale yellow, strongly odoriferous flowers Kawano & Noguchi 1973, Hotta et al. 1984, that are nocturnal or extended (night and day) Hasegawa et al. 2006, Nitta et al. 2007, Yahara flowering. AlthoughH. hakuunensis and H. major 2007). Hemerocallis sect. Capitatae and subclus- were classified in H. sect. Fulvae by Matsuoka & ter [B], the latter a diploid group of H. sect. Ful- Hotta (1966) and Hotta (2016), they have pale vae (Hotta 2016), were represented long branches orangish yellow, usually faintly odoriferous flow- with relatively little uncertainty, indicating well- ers in contrast with the species with more or less differentiated lineages. The evidence suggests reddish flowers without odor of H. sect. Fulvae. that introgressions are nearly all in one direction, Additionally, Hemerocallis sect. Fulvae usually but further plastid DNA analysis is needed to as- has horizontally spreading rhizomes and a tawny certain this suggestion. V-shaped blotch at the base of the inner tepals, while H. sect. Hemerocallis lacks them. Although Species or variety Matsuoka & Hotta (1966) and Hotta (2016) no- Cluster I exactly corresponds to Hemerocallis ticed that H. hakuunensis and H. major lacked the middendorffii sensu Hotta (2016). In this cluster, spreading rhizomes and V-shaped blotch on the H. middendorffii var. middendorffii (13) is isolat- tepals, they nevertheless classified the two species ed from subcluster [A], in which H. middendorffii in H. sect. Fulvae, placing more weight on the few var. esculenta (14–17) and var. exaltata (18–26) characteristics of the flowers, such as orangish ba- were loosely separated without mixing. Thus, the sic color, diurnal flowering, and lack of strong recognition of the three varieties within H. mid- odor, although even these characteristics of the dendorffiiis supported. In addition to plants from two species are somewhat different from those of Tobishima in Yamagata Prefecture, Hotta (2016) other members of H. sect. Fulvae, as mentioned included plants from Akita Prefecture and Sado above. Island in Niigata Prefecture in H. middendorffii In our findings using the MIG-seq data (Fig. var. exaltata. Our study supports Hotta’s circum- 2), Hemerocallis hakuunensis and H. major (sect. scription of var. exaltata because samples of var. Fulvae) were clustered not with the other mem- exaltata, from Akita Prefecture (18) and Sado Is- bers of H. sect. Fulvae, but with members of H. land (24–26), did not form mixed clusters with sect. Hemerocallis. Thus, we transfer H. hakuu- var. esculenta. In var. exaltata, five samples from nensis and H. major from H. sect. Fulvae to H. Tobishima Isl. (19–23) and three samples from sect. Hemerocallis. Consequently, H. sect. Fulvae Sado Isl. (24–26) were grouped, but a sample is confined to species with more or less reddish from Akita Prefecture (18) was excluded from the flowers and absence of odor, while H. sect. Hem- two groups. The grouping indicates that gene erocallis includes species with strongly fragrant flow between the three islands (Honshu, Sado Isl. lemon flowers to pale orangish yellow, faintly fra- and Tobishima) is rather limited. grant (rarely nearly imperceptibly fragrant) flow- Cluster II exactly agrees to Hemerocallis ful- February 2020 Murakami & al.—Sections of Hemerocallis Recircumscribed by MIG-seq 7 va sensu Hotta (2016). In cluster II, the diploid [D], although the sample of var. citrina diverged varieties of H. fulva, i.e. var. disticha (30–33), var. from the four samples of var. vespertina at the littorea (38, 39) and var. longituba (40), were in base of the subcluster [D]. Thus, the MIG-seq subcluster [B], while the triploid varieties of H. data confirmed that vars. citrina and vespertina fulva, i.e. var. fulva (27–29), var. kwanso (34–36) are conspecific, but differ at the infraspecific lev- and var. sempervirens (41), were in subcluster el (which we recognize at the varietal level). The [C]. Thus, it may be that the three triploid variet- hypothesis of multiple origins of the Japanese H. ies have the same origin. Subcluster [C], however, citrina var. vespertina proposed by Noguchi & was rather loose, an average pairwise genetic dis- Hong (2004) was not confirmed in the present tance of 0.195 compared to other subclusters, study. Three samples of H. minor (10–12) were which ranged from 0.095 to 0.159 (Table 2). Fur- clustered into one and embedded in H. lilioaspho- ther, the triploid samples were genotyped as dip- delus. The separation of H. lilioasphodelus and loids in the settings of Stacks v.1.4.4 in this study. H. minor at the species level is questionable. Hot- Thus, it may be that the formation of subcluster ta (in Matsuoka & Hotta, 1966) once treated H. [C] is the product of an error. The coherence of minor as a variety of H. flava (= H. lilioasphode- subcluster [C] should be checked by a different lus). Our study indicates that Hotta’s hypothesis method, such as plastid DNA analysis. In the dip- is worth reevaluating. A new combination, H. lil- loid subcluster [B], two samples of H. fulva var. ioasphodelus L. var. minor (Mill.) M. N. Tamura, littorea (38, 39) were embedded in H. fulva var. is therefore proposed in this study. Xiong et al. disticha, which is widely distributed in grass- (1996) arrived as the same conclusion, but recog- lands from Japan to the Himalaya. Thus, H. fulva nized var. minor at the subspecific level. Because var. littorea, which is a coastal form along the Pa- H. lilioasphodelus var. thunbergii was nested cific Ocean from the Kanto District to Kyushu, within H. lilioasphodelus var. lilioasphodelus, may be derived from var. disticha. Only one sam- we reduce var. thunbergii to synonymy under the ple of H. fulva var. littorea from the Miura Penin- latter. Hemerocallis lilioasphodelus var. minor sula, Kanagawa Prefecture (37), was included in can be considered an extreme form (smaller, with the triploid subcluster, although H. fulva var. lit- slender roots, narrow grass-like leaves, shorter torea should be a diploid based on Hotta’s (2016) scape and fewer flowers) derived from the main findings. stock of H. lilioasphodelus. Kitamura (1964) recognized two species in The sample (6) of Hemerocallis citrina var. cluster II: Hemerocallis fulva (including ‘disti- vespertina from Tsushima in Nagasaki Prefec- cha’, ‘fulva’, ‘kwanso’ and ‘longituba’ in this ture was clustered with three samples of H. study) and H. aurantiaca (including ‘littore’ and hakuunensis (42–44). As Hotta et al. (1984) sug- ‘major’ in this study). However, subcluster [B] gested, a high possibility of introgressive hybrid- (‘disticha’, ‘littorea’ and ‘longituba’) is an admix- ization between H. citrina var. vespertina and H. ture of Kitamura’s two species. Further, part of hakuunensis on exists on Tsushima. The varia- Kitamura’s H. aurantiaca (‘major’) was not in- tion in flowering in the populations included in cluded in the II, but in cluster III. Thus, Kitamu- our samples (6) in subcluster [F] of H. hakuunen- ra’s species concept concerning H. fulva and H. sis might be explained by introgression. This aurantiaca is not supported based on our MIG- seems plausible, because the flowers of hybrids seq data. between H. citrina var. vespertina and H. hakuu- In cluster III, four samples of Hemerocallis nensis on Tsushima often do not show intermedi- citrina var. vespertina from Yamanashi, Kyoto ate traits, but instead resemble the flowers of pure and Wakayama prefectures (2–5) and one sample H. citrina var. vespertina (Yahara 2007). of H. citrina var. citrina (1) were further clustered 8 Acta Phytotax. Geobot. Vol. 71

Key to the sections of Hemerocallis from Japan and adjacent regions

1a. Flowers lemon, pale yellow or orangish yellow, without V-shaped blotch at base, nocturnal, diurnal, or extended (night and day) flowering, with strong or faint (or without) odor; horizontally spreading rhizomes absent...... 2 1b. Flowers orange or red, with tawny V-shaped blotch at base of inner tepals, diurnal, without odor; horizontally spreading rhizomes present ...... H. sect. Fulvae 2a. Inflorescence compact; sterile bracts on scape absent; flowers orangish yellow, diurnal or extended (night and day) flowering, with faint odor ...... H. sect. Capitatae 2b. Inflorescence loose; sterile bracts on scape present; flowers lemon, pale yellow or pale orangish yellow, nocturnal, diurnal or extended (night and day) flowering, with strong or faint (or no) odor ...... H. sect. Hemerocallis

Taxonomic Change References

Hemerocallis lilioasphodelus L. var. minor Bailey, L. H. 1930. Hemerocallis: the day-lilies. Gentes (Mill.) M. N. Tamura, nov. Herb. 2: 143–156. Bryant, D. & V. Moulton. 2004. Neighbor-Net: an agglom- Basionym: Hemerocallis minor Mill., Gard. erative method for the construction of phylogenetic Dict., ed. 8. Hemerocallis no. 2 (1768). networks. Molec. Biol. Evol. 21: 255–265. Hemerocallis flava L. var. minor (Mill.) M. Catchen, J., P. A. Hohenlohe, S. Bassham, A. Angel & W. Hotta in Acta Phytotax. Geobot. 22: 40 (1966). A. Cresko. 2013. Stacks: an analysis tool set for popu- Hemerocallis lilioasphodelus L. subsp. minor lation genomics. Molec. Ecol. 22: 3124–3140. Chen, X. Q. & J. Noguchi. 2000. Hemerocallis. In: Wu, Z. (Mill.) Z. T. Xiong, Acta Phytotax. Sin. 34: 590 Y. & P. H. Raven (eds.), Flora of China, vol. 24, pp. (1996). 161–165. Science Press, Beijing, and Missouri Botani- cal Garden Press, St. Louis. Classification of taxa of Hemerocallis Doyle, J. J. & J. L. Doyle. 1987. A rapid DNA isolation pro- examined in this study cedure for small quantities of fresh tissue. Phyto- chem. Bull. 19: 11–15. Hasegawa, M., T. Yahara, A. Yasumoto & M. Hotta. 2006. H. sect. Hemerocallis: H. citrina var. citrina, Bimodal distribution of flowering time in a natural H. citrina var. vespertina, H. hakuunensis, H. lil- hybrid population of (Hemerocallis fulva) and ioasphodelus var. lilioasphodelus (= H. lilioas- nightlily (Hemerocallis citrina). J. Res. 119: 63- phodelus var. thunbergii), H. lilioasphodelus var. 68. Hotta, M. 2016. Hemerocallis. In: Iwatsuki, K., D. E. minor (= H. minor) and H. major Boufford & H. Ohba (eds.), Flora of Japan, vol. IVb. H. sect. Capitatae: H. middendorffii var. mid- Angiospermae: Monocotyledoneae (b), pp. 130–134. dendorffii, H. middendorffii var. esculenta and H. Kodansha, Tokyo. middendorffii var. exaltata Hotta, M, M. Ito & I. Okada. 1984. Anthesis of the genus H. sect. Fulvae: H. fulva var. fulva, H. fulva Hemerocallis and its variation. Special mentions to nocturnal H. thunbergii of Tsushima & Hirado Is- var. disticha, H. fulva var. kwanso, H. fulva var. lands, Western Japan. Acta Phytotax. Geobot. 35: 84– littorea, H. fulva var. longituba and H. fulva var. 93 (in Japanese). sempervirens Hu, S. Y. 1969. A new daylily species from Sakhalin Is- land. Hemerocallis J. 23: 42–43. Huson, D. H. & D. Bryant. 2006. Application of phyloge- We express our sincere gratitude to Dr. Kazuhiko Haya- netic networks in evolutionary studies. Molec. Biol. shi, Mr. Jiro Oda, Mr. Yujiro Horii, Mr. Gen Ito and Mr. Evol. 23: 254–267. Seita T. Watanabe for supplying plant materials. Our Kawano, S. & J. Noguchi. 1973. Biosystematic studies on thanks are due to Prof. Hidetoshi Nagamasu for nomen- the genus Hemerocallis (Liliaceae). J. Coll. Lib. Arts. clatural discussion This study was supported in part by Toyama Univ. 6: 111–137. ISHIZUE 2019 of Kyoto University Research Develop- Kitamura, S. 1964. Liliaceae. In: Kitamura, S., G. Murata ment Program (to M. N. Tamura). & T. Koyama (eds.), Coloured Illustrations of Herba- ceous Plants of Japan (Monocotyledoneae), pp. 90– 157. Hoikusha, Osaka (in Japanese). February 2020 Murakami & al.—Sections of Hemerocallis Recircumscribed by MIG-seq 9

Lassmann, T., Y. Hayashizaki & C. O. Daub. 2009. Tag- Noguchi, J. & D.-Y. Hong. 2004. Multiple origins of the Dust—a program to eliminate artifacts from next gen- Japanese nocturnal Hemerocallis citrina var. vesper- eration sequencing data. Bioinformatics 25: 2839– tina (: Hemerocallidaceae): evidence 2840. from noncoding chloroplast DNA sequences and mor- Matsuoka, M. & M. Hotta. 1966. Classification of Hem- phology. Int. J. Plant Sci. 165: 219–230. erocallis in Japan and its vicinity. Acta Phytotax. Suyama, Y. & Y. Matsuki. 2015. MIG-seq: an effective Geobot. 22: 25–43 (in Japanese with English key to PCR-based method for genome-wide single-nucleo- the sections and species and English taxonomic treat- tide polymorphism genotyping using the next-genera- ments). tion sequencing platform. Sci. Rep. 5: 16963. Nakai, T. 1932. Hemerocallis Japonica. Bot. Mag. (To- Webb, D. A. 1980. Hemerocallis. In: Tutin, T. G., V. H. kyo) 46: 111–123. Heywood, N. A. Burges, D. M. Moore, D. H. Valen- Nakao, S. & K. Yamashita. 1956. Variation in some plant tine, S. M. Walters & D. A. Webb (eds.), Flora Euro- population. In: Komai, T. & K. Sasaki (eds.), Shudan paea, vol. 5, 19. Cambridge University Press, London. Idengaku (Population Genetics), pp. 249–254. Baifu- Xiong, Z. T., S. C. Chen & D. Y. Hong. 1996. Taxonomic kan, Tokyo (in Japanese). studies in the nocturnal flowering group of Hemero- Nitta, K., M. Hasegawa, T. Miyake, A. A. Yasumoto and callis (Liliaceae) from China. Acta Phytotax. Sin. 34: T. Yahara. 2007. Genetic basis of floral traits contrib- 586–591. uting to pollination syndromes of nightlily (Hemero- Yahara, T. 2007. Exploring the genetic backgrounds of callis citrina) and daylily (H. fulva). Jap. J. Ecol. 57: differentiation in floral scent and color between 100–106 (in Japanese). daylily and nightlily. Bunrui 7: 93–110 (in Japanese).

Received June 6, 2019; accepted September 6, 2019 10 Acta Phytotax. Geobot. Vol. 71 15 0.37788018 0.40074906 0.40555556 0.41397849 0.46594005 0.14728682 0.12478921 0.12714777 0.10874200 0.14011299 0.14011299 0.13001215 0.35338346 0.12500000 0.37453184 0.13951012 0.37847222 0.12359551 0.35510204 0.12500000 0.40191388 0.09779951 0.36444444 0.40000000 0.45631068 0.41784038 0.41104294 0.41104294 0.42622951 0.45945946 0.44915254 0.40092166 14 0.45026178 0.42325581 0.34210526 0.42528736 0.47297297 0.08484848 0.10053860 0.09982175 0.07535642 0.10025063 0.08010336 0.37401575 0.09815242 0.41125541 0.41125541 0.10532407 0.34873950 0.09152542 0.36923077 0.11851852 0.11851852 0.37894737 0.10928144 0.37688442 0.13731343 0.43956044 0.46441948 0.38068182 0.41608392 0.39215686 0.43352601 0.51020408 0.42131980 13 0.39062500 0.37096774 0.37333333 0.41463415 0.41216216 0.10869565 0.12948207 0.13610586 0.11286682 0.11286682 0.15047880 0.12309820 0.35059761 0.12944162 0.38362069 0.14444444 0.38864629 0.11150442 0.11150442 0.39062500 0.14262023 0.35638298 0.14012739 0.34653465 0.14405360 0.40796020 0.12436975 0.45421245 0.39226519 0.39100346 0.31446541 0.37714286 0.46896552 0.41463415 12 0.36263736 0.35071090 0.33333333 0.35403727 0.40239044 0.33928571 0.36813187 0.35454545 0.34375000 0.41639344 0.38795987 0.25563910 0.41486068 0.17933131 0.41955836 0.19016393 0.36868687 0.24454148 0.36480687 0.35028249 0.38582677 0.34375000 0.35887097 0.36111111 0.36111111 0.37037037 0.37288136 0.39748954 0.35000000 0.34959350 0.34591195 0.34591195 0.34693878 0.42745098 0.38674033 11 0.36419753 0.37500000 0.37323944 0.35849057 0.37551020 0.34939759 0.38172043 0.36199095 0.34693878 0.42811502 0.42811502 0.38205980 0.24701195 0.24701195 0.39506173 0.20845921 0.39240506 0.21974522 0.33009709 0.22746781 0.39430894 0.36619718 0.36032389 0.35802469 0.34538153 0.35502959 0.36073059 0.41904762 0.33877551 0.41481481 0.08108108 0.37826087 0.27814570 0.33757962 0.35627530 0.37575758 10 0.43750000 0.45454545 0.42131980 0.40758294 0.45316456 0.38775510 0.39183673 0.38345865 0.39090909 0.41125541 0.41125541 0.39819005 0.29916898 0.40704501 0.21263158 0.40495868 0.21719457 0.35294118 0.35294118 0.20679887 0.39000000 0.41904762 0.40287770 0.37735849 0.41052632 0.43636364 0.38888889 0.41297935 0.40752351 0.39810427 0.08750000 0.38950276 0.11155378 0.11155378 0.39459459 0.41628959 0.45063291 0.37500000 9 0.40909091 0.42677824 0.35849057 0.37837838 0.45098039 0.39603960 0.37229437 0.35655738 0.37442922 0.40446650 0.40157480 0.32371795 0.39371981 0.18867925 0.39860140 0.22507123 0.34496124 0.25631769 0.41639344 0.36842105 0.36666667 0.36842105 0.38095238 0.35514019 0.36267606 0.44656489 0.38775510 0.40243902 0.10873440 0.38741722 0.12016293 0.37142857 0.13186813 0.39560440 0.41914191 0.39500000 8 0.43666667 0.41558442 0.41911765 0.41911765 0.40495868 0.43902439 0.46206897 0.41860465 0.41457286 0.39099526 0.39755352 0.40400000 0.38320611 0.38320611 0.35844156 0.39298670 0.22993492 0.39625000 0.26849894 0.37701149 0.37701149 0.28232190 0.39329806 0.39298246 0.39144737 0.40000000 0.37455830 0.43081761 0.37111111 0.37111111 0.43723849 0.36000000 0.37931034 0.13903743 0.41818182 0.12994350 0.39406780 0.19975186 0.20272315 0.46153846 0.43579767 7 0.35111111 0.35111111 0.38983051 0.41503268 0.36458333 0.40703518 0.41734417 0.36956522 0.39542484 0.38412698 0.39552239 0.42600000 0.39630390 0.32267442 0.40145985 0.23711340 0.23711340 0.39278937 0.26618705 0.38000000 0.23493976 0.40476190 0.38116592 0.38116592 0.38793103 0.38495575 0.37226277 0.37837838 0.41525424 0.41587302 0.37398374 0.38725490 0.14549180 0.40114613 0.40114613 0.12153518 0.34825871 0.15821813 0.15601504 0.39303483 0.18668407 0.38781163 0.38781163 6 0.34158416 0.32460733 0.32661290 0.35897436 0.31645570 0.40522876 0.35960591 0.36683417 0.35135135 0.39795918 0.38208955 0.35646688 0.23728814 0.37700535 0.14285714 0.37533512 0.16397849 0.34033613 0.12654321 0.36700337 0.32275132 0.36949153 0.32620321 0.36206897 0.40860215 0.35907336 0.40229885 0.37735849 0.35326087 0.16315789 0.33571429 0.17468354 0.34078212 0.20841300 0.16816143 0.34848485 0.25572519 0.20046083 0.39935065 5 0.35751295 0.39361702 0.38135593 0.35542169 0.39459459 0.42198582 0.40414508 0.43127962 0.35294118 0.35294118 0.40860215 0.45345345 0.43708609 0.29794521 0.43093923 0.19638243 0.43567251 0.22067039 0.41004184 0.22183099 0.41379310 0.34183673 0.42446043 0.35751295 0.43103448 0.40594059 0.38775510 0.41603053 0.39382239 0.37430168 0.15384615 0.39024390 0.18781726 0.36477987 0.18853974 0.19650655 0.36956522 0.27854671 0.25210084 0.37662338 0.14449541 4 0.41558442 0.37500000 0.42525773 0.40265487 0.41153846 0.41153846 0.42602496 0.42176871 0.41916168 0.41242938 0.40549828 0.43956044 0.42056075 0.30000000 0.41463415 0.20866142 0.43913043 0.26274510 0.41129032 0.41129032 0.27696078 0.39495798 0.33333333 0.41448692 0.36140351 0.41753653 0.38019169 0.41075795 0.42535787 0.39225182 0.37109375 0.16555184 0.39793814 0.17477477 0.38053097 0.18810680 0.20116618 0.20116618 0.37455830 0.25275827 0.23044693 0.40804598 0.15294118 0.15294118 0.16746411 3 0.37373737 0.31794872 0.36086957 0.34415584 0.42073171 0.38095238 0.35714286 0.39393939 0.38400000 0.40178571 0.45179063 0.41818182 0.28424658 0.41909814 0.22437673 0.45428571 0.22126437 0.40000000 0.22924901 0.39285714 0.37142857 0.34113712 0.34113712 0.37853107 0.37132353 0.41062802 0.39607843 0.39043825 0.41635688 0.36363636 0.17206478 0.37500000 0.19257541 0.35714286 0.21388368 0.21669980 0.31645570 0.25694444 0.23245614 0.42164179 0.14159292 0.11839323 0.18779343 2 0.30136986 0.31481481 0.35483871 0.35820896 0.37500000 0.37610619 0.35714286 0.38728324 0.36150235 0.40099010 0.42715232 0.40225564 0.26446281 0.36824324 0.18987342 0.41496599 0.22027972 0.37988827 0.20465116 0.20465116 0.37554585 0.29696970 0.36752137 0.33783784 0.35146444 0.36875000 0.34862385 0.36923077 0.37398374 0.29577465 0.18269231 0.36966825 0.21229050 0.36363636 0.21342926 0.23222749 0.36879433 0.26269316 0.23469388 0.38118812 0.38118812 0.03758170 0.15427509 0.14420804 0.18750000 1 0.38435374 0.40344828 0.41340782 0.42982456 0.47058824 0.43333333 0.41134752 0.41134752 0.41269841 0.39823009 0.40225564 0.44850498 0.43025540 0.30861244 0.42627737 0.22485207 0.43981481 0.25879917 0.40909091 0.28643216 0.40193705 0.37453184 0.42173913 0.38800000 0.42231947 0.45454545 0.40860215 0.44239631 0.43007916 0.40476190 0.17733090 0.41176471 0.41176471 0.19760479 0.40000000 0.22415940 0.23555556 0.41281139 0.41281139 0.26867220 0.25000000 0.40322581 0.20030120 0.20159681 0.22617124 0.21499548 0.19831933 9 8 7 5 2 3 4 6 32 31 30 29 28 27 26 25 24 23 22 21 45 11 11 20 44 19 43 18 42 17 41 16 40 15 39 14 38 13 37 12 36 35 10 34 33 Samples Appendix Pairwise 1. genetic distances between samples the of genus Hemerocallis from Japan. The sample numbers correspond to 1. those Table of February 2020 Murakami & al.—Sections of Hemerocallis Recircumscribed by MIG-seq 11 30 0.37254902 0.36915888 0.36186770 0.35655738 0.32403433 0.10204082 0.08660352 0.10185185 0.35111111 0.35111111 0.32369942 0.32380952 0.32826087 0.09393939 0.07919463 0.08998549 29 44 0.35779817 0.37241379 0.33142857 0.37106918 0.22432432 0.29780564 0.27401130 0.27401130 0.33014354 0.25757576 0.20212766 0.13500000 0.15763547 0.32853717 0.32044199 0.29203540 0.31007752 0.31007752 0.24731183 0.24731183 43 28 0.40833333 0.39884393 0.39285714 0.38297872 0.27249357 0.33237822 0.31232092 0.36725664 0.29866667 0.23420074 0.08530806 0.09484536 0.35434783 0.35372340 0.30517711 0.30517711 0.33096927 0.12600536 0.12600536 0.23964497 0.16966581 0.16966581 42 27 0.37078652 0.39215686 0.42553191 0.40672783 0.29629630 0.32150313 0.35489510 0.37807606 0.31467181 0.25306577 0.12028725 0.11235955 0.11235955 0.37239869 0.37543253 0.32656827 0.35792350 0.35792350 0.15289256 0.15289256 0.21725240 0.21725240 0.14137931 0.14137931 0.16129032 0.16129032 0.10962567 0.10962567 41 26 0.40000000 0.36040609 0.37755102 0.44370861 0.40909091 0.33750000 0.48091603 0.46798030 0.38461538 0.39814815 0.41071429 0.43065693 0.48148148 0.42253521 0.42335766 0.39884393 0.39884393 0.31932773 0.31932773 0.37401575 0.37401575 0.40714286 0.40714286 0.35542169 0.35542169 0.42857143 0.42857143 0.34196891 0.34196891 0.37714286 0.37714286 25 40 0.36073059 0.37810945 0.40540541 0.40782123 0.41216216 0.40000000 0.44366197 0.48068670 0.42361111 0.42361111 0.41596639 0.45714286 0.42483660 0.49789030 0.43670886 0.40718563 0.43333333 0.43333333 0.40689655 0.33204633 0.40689655 0.32000000 0.44583333 0.44583333 0.31606218 0.31606218 0.04850746 0.39673913 0.39673913 0.30303030 39 24 0.34703196 0.38500000 0.39189189 0.37662338 0.35185185 0.36633663 0.39664804 0.46122449 0.40127389 0.40154440 0.41176471 0.41176471 0.45070423 0.45454545 0.42196532 0.38922156 0.41395349 0.41395349 0.35000000 0.33596838 0.33596838 0.41780822 0.41780822 0.36000000 0.41897233 0.41897233 0.33658537 0.33658537 0.03441296 0.03441296 0.03703704 0.03703704 0.35828877 0.35828877 0.28292683 0.11801242 0.11801242 38 23 0.36158192 0.38554217 0.37430168 0.40277778 0.42253521 0.42038217 0.42307692 0.41666667 0.44029851 0.46632124 0.37500000 0.45736434 0.50243902 0.40909091 0.47761194 0.47761194 0.44186047 0.44186047 0.43750000 0.39782016 0.39782016 0.43902439 0.43902439 0.43946188 0.43946188 0.46268657 0.38831615 0.38831615 0.05989583 0.05989583 0.06419753 0.06419753 0.37722420 0.06391753 0.06391753 0.33333333 0.33333333 0.12903226 0.12903226 0.05903188 0.05903188 37 22 0.42414861 0.42474916 0.40575080 0.40377358 0.45149254 0.44905660 0.45848375 0.49029126 0.46987952 0.43378995 0.42647059 0.45147679 0.51162791 0.51162791 0.47272727 0.47177419 0.48126801 0.48126801 0.43010753 0.43010753 0.38559322 0.38559322 0.44954128 0.44954128 0.35119048 0.35119048 0.48611111 0.48611111 0.35828877 0.35828877 0.06576728 0.06576728 0.07580175 0.07580175 0.38547486 0.38547486 0.08807588 0.01762115 0.01762115 0.05095541 0.05095541 0.33240997 0.34224599 0.37072243 0.37072243 36 21 0.38644068 0.41403509 0.40490798 0.38043478 0.40316206 0.39759036 0.43629344 0.47355164 0.43043478 0.39370079 0.37745098 0.43600000 0.48500000 0.47200000 0.43307087 0.43384615 0.43384615 0.35960591 0.35960591 0.35869565 0.35869565 0.40528634 0.35833333 0.35833333 0.44047619 0.44047619 0.35423197 0.35423197 0.07692308 0.08071749 0.08071749 0.40213523 0.40213523 0.09028728 0.13301282 0.13301282 0.04455446 0.28456914 0.28456914 0.04506066 0.31794872 0.31794872 0.34897025 0.15055468 0.15055468 35 20 0.41828255 0.42633229 0.38770053 0.39102564 0.41454545 0.45323741 0.47619048 0.48407643 0.41886792 0.42031873 0.40888889 0.43866171 0.51968504 0.42372881 0.43598616 0.45251397 0.45251397 0.38571429 0.38571429 0.37327189 0.37327189 0.42911877 0.42911877 0.37419355 0.37419355 0.46224678 0.39893617 0.39893617 0.07187500 0.07187500 0.08458390 0.41764706 0.41764706 0.08827404 0.26385224 0.06112853 0.06112853 0.29652997 0.05960265 0.29947917 0.29947917 0.05916667 0.05916667 0.36697248 0.30681818 0.30681818 0.25000000 19 34 0.39142857 0.42507645 0.42228739 0.41139241 0.41139241 0.44402985 0.46125461 0.49134948 0.48728814 0.45149254 0.44785276 0.44796380 0.46718147 0.53444676 0.49480969 0.45907473 0.45552561 0.45552561 0.40865385 0.38009050 0.41666667 0.41666667 0.37426901 0.37426901 0.46828358 0.46828358 0.41919192 0.41919192 0.09621451 0.09621451 0.09699454 0.41621622 0.41621622 0.10574413 0.10574413 0.29156010 0.29156010 0.05520505 0.05520505 0.33333333 0.33333333 0.06119403 0.06119403 0.30946292 0.05727452 0.05727452 0.06219709 0.37037037 0.37037037 0.30256410 0.30256410 0.00514580 0.00514580 0.27194492 33 18 0.37606838 0.39639640 0.40178571 0.36190476 0.40828402 0.37113402 0.37113402 0.44508671 0.43511450 0.43511450 0.41176471 0.41176471 0.44814815 0.42758621 0.41666667 0.48188406 0.43147208 0.46073298 0.39035088 0.33566434 0.38860104 0.38860104 0.41666667 0.41666667 0.38281250 0.38281250 0.43086817 0.43086817 0.37540453 0.37540453 0.08134921 0.08134921 0.09073724 0.35873016 0.35873016 0.09505703 0.09505703 0.35338346 0.35338346 0.08252427 0.12893082 0.12893082 0.10583446 0.10583446 0.09681529 0.09681529 0.11945813 0.11945813 0.09557945 0.08448753 0.10367893 0.10367893 0.36346154 0.36346154 0.34879406 0.32413793 0.33179191 32 17 0.39051095 0.35507246 0.36271186 0.36271186 0.36734694 0.36363636 0.39819005 0.41624365 0.46518987 0.36500000 0.38066465 0.41717791 0.45098039 0.48000000 0.40000000 0.43925234 0.41132075 0.41132075 0.35260116 0.35260116 0.36071429 0.36071429 0.39037433 0.39037433 0.32105263 0.32105263 0.44473684 0.34433962 0.34433962 0.09833024 0.13949580 0.13949580 0.36764706 0.36764706 0.11304348 0.11304348 0.32266010 0.32266010 0.10917031 0.10917031 0.14003945 0.14003945 0.13101904 0.13101904 0.07413011 0.07413011 0.13117284 0.13117284 0.12696747 0.10266940 0.11177885 0.07102593 0.07102593 0.35588972 0.35588972 0.11231884 0.11231884 0.34577114 0.32822086 0.30834753 16 31 0.38698630 0.37588652 0.36842105 0.40370370 0.41706161 0.35897436 0.42790698 0.47398844 0.43750000 0.42696629 0.41145833 0.41145833 0.44776119 0.44776119 0.49725275 0.43750000 0.42794760 0.45307443 0.45307443 0.40526316 0.40526316 0.37269373 0.37269373 0.47150259 0.47150259 0.33734940 0.47567568 0.47567568 0.36018957 0.36018957 0.11363636 0.11363636 0.13500000 0.35643564 0.11831442 0.11831442 0.29925187 0.29925187 0.08367347 0.08367347 0.13871636 0.13871636 0.12715517 0.12715517 0.07836457 0.07836457 0.11516315 0.11516315 0.08680143 0.08680143 0.12229102 0.13627451 0.11123348 0.08655126 0.08655126 0.32225064 0.30120482 0.08333333 0.08333333 0.12236842 0.32085561 0.32404181 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 45 28 44 27 43 26 25 42 24 41 23 40 22 39 20 17 18 19 21 38 37 35 32 33 34 36 Samples Samples Appendix continued. 1. Appendix continued. 1.