Analysis and evaluation of morphological and molecular polymorphism in the hybridization of <i>Elaeagnus ×maritima</i> and <i>E. ×submacrophylla</i>

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적 요:

녹보리똥나무와 큰보리장나무는 형태적 특성에 기반하여 잡종 분류군으로 제안된 바 있으나, 이에 대한 분류학적 실체가 불명확하다. 본 연구에서는 녹보리똥나무와 큰보리장나무의 잡종 기원을 밝히기 위하 여 현장 조사와 표본관에 소장된 표본을 검토하여 형태적 특징을 관찰하였으며, 핵리보솜 구간(internal transcribed spacer, 5S non-transcribed spacer)과 엽록체 구간(matK)의 염기서열을 비교·분석하였다. 형태적 특징을 관찰한 결과, 녹보리똥나무는 보리장나무와, 큰보리똥나무는 통영볼레나무와 형태적으로 유사성을 보였으나, 분자 분석 결과, 녹보리똥나무는 핵리보솜 구간에서 보리장나무와 보리밥나무의 서열의 혼성화가 관찰되었다. 큰보리장나무는 다양한 양상이 관찰되었는데, 일부 개체는 핵리보솜 구간에서 통영볼레나무와 보리밥나무의 서열의 혼성화가, 엽록체 구간에서는 보리밥나무 서열이 관찰되었다. 다른 개체는 핵리보솜 구간 에서는 보리밥나무의 서열을 보였으나, 엽록체 구간에서는 통영볼레나무의 서열이 관찰되어 핵과 엽록체간 불일치를 보였다. 일부 개체는 통영볼레나무와 보리밥나무의 중간형을 보였으나, 핵리보솜과 엽록체 구간 모두 보리밥나무 서열이 관찰되었다. 이러한 결과는 두 종이 잡종기원이며, 부모종 또는 잡종 개체간 교배가 빈번히 일어나는 것을 시사한다.

주요어: 보리수나무속, 엽록체 DNA, 유전적 다양성, 잡종, 핵리보솜 DNA, 형태적 특징

Abstract

The taxonomic identity of Elaeagnus ×maritima and E. ×submacrophylla (Elaeagnaceae) in Korea is unclear, yet they are presumed to be hybrid taxa based on their morphology. To determine their hybrid origins, a morphological analysis (field surveys and specimen examinations) and a molecular analysis involving two nuclear ribosomal DNA (nrDNA) regions (internal transcribed spacer and 5S non-transcribed spacer) and one chloroplast DNA (cpDNA) region (matK) were conducted. The morphological analysis revealed that E. ×maritima showed certain morphological similarities to E. glabra, whereas E. ×submacrophylla showed certain morphological similarities to E. pungens. However, the molecular analysis indicated that E. ×maritima exhibited additive species-specific sites of E. glabra and E. macrophylla in the nrDNA regions. Notably, E. ×submacrophylla showed various aspects, with some individuals exhibiting additive species-specific sites of E. pungens and E. macrophylla in the nrDNA and E. macrophylla sequences in the cpDNA regions, some individuals exhibiting E. macrophylla sequences in the nrDNA and E. pungens sequences in the cpDNA regions, and some individuals displaying E. macrophylla sequences in both the nrDNA and cpDNA regions, despite an intermediate morphology between E. pungens and E. macrophylla. These results indicate that these two species are of hybrid origin and frequently cross between parental and hybrid individuals.

Keywords: chloroplast DNA, Elaeagnus, hybridization, molecular polymorphism, morphological traits, nuclear ribosomal DNA

INTRODUCTION

The genus Elaeagnus L. belongs to the Elaeagnaceae family and comprises approximately 90 species distributed from eastern Asia to Queensland in northeastern Australia, with a few species even occurring in southern Europe and North America (Qin and Gilbert, 2007). Elaeagnus species are characterized by silvery white or reddish brown peltate scales on most parts of the shoot, alternate leaves, bisexual flowers, 4-lobed calyx, epipetalous, antisepalous, 4-stamens, and 8-riped seed.

The genus Elaeagnus has traditionally been divided into two sections: Sempervirentes Serv., which is evergreen, flowering in autumn and fruiting in spring: and Elaeagnus, which is deciduous, flowering in spring and fruiting in autumn (Servettaz, 1909).

Within the section Sempervirentes in Korea, five taxa (E. glabra Thunb., E. ×maritima Koidz., E. macrophylla Thunb., E. ×submacrophylla Serv., and E. pungens Thunb.) have been identified. Among these, E. ×maritima and E. ×submacrophylla have been proposed as hybrid taxa based on their morphological characteristics (Ohba, 1999; Ko, 2015). Elaeagnus ×maritima was initially considered as a hybrid between E. glabra and E. macrophylla (Ohba, 1999), whereas Ki (2004) and Koh (2005) regarded it as a synonym of E. glabra based on a comparison of external morphology, trichomes, and pollen. Similarly, E. ×submacrophylla is regarded as a hybrid between E. pungens and E. macrophylla (Servettaz, 1909; Ohba, 1999), but Lee (1996) proposed that it is a hybrid between E. glabra and E. macrophylla instead. On the other hand, while E. ×nikaii Nakai, reported on Mt. Mireuksan (Tongyeong, Korea), was regarded as a hybrid between E. pungens and E. macrophylla (Nakai, 1928), no further research has been conducted on individuals in Mt. Mireuksan.

In the taxonomic classification of species within section Sempervirentes in Korea, a consensus among researchers has not yet been reached. This is because of the high morphological variation observed in this group, as well as the possibility of hybridization, which makes species identification challenging. To gain a better understanding of the taxonomic identity of this group of species, it is crucial to genetically examine putative hybrids to determine whether hybridization occurs. Molecular markers such as nuclear ribosomal DNA (nrDNA) regions have proven to be valuable tools for identifying hybrid taxa progenitors because they exhibit biparental inheritance (Sang et al., 1995; Li, 2006; Du et al., 2009; Les et al., 2009; Hřibová et al., 2011; Kokubugata et al., 2011). The detection of parental genomes in putative hybrid taxa using these markers can provide direct evidence of hybrid speciation. Additionally, the analysis of chloroplast DNA (cpDNA) data can aid in the detection and determination of the direction of hybridization in plants (Rieseberg et al., 1993; Schwarzbach and Rieseberg, 2002).

In this study, the taxonomic identity of putative hybrid taxa was examined by observing their morphological traits, which were compared with the original descriptions, type specimens, and collected and stored specimens. Additionally, two nrDNA regions (internal transcribed spacer [ITS] and 5S non-transcribed spacer [NTS]) and one cpDNA region (matK) were analyzed. Detailed descriptions, illustrations, and photographs were prepared to provide comprehensive documentation of the findings.

MATERIALS AND METHODS

Morphological observation

To compare the morphological characteristics of the putative hybrids and their closely related taxa, we collected specimens from the field sampling in five localities in Korea (Fig. 1) and deposited them in the herbarium of the Korea National Arboretum (KH). Additional morphological examinations were conducted by observing specimens from the herbariums of the KH, National Institute of Biological Resources (KB), Sungkyunkwan University (SKK), and Yeungnam University (YNUH). Type specimens were obtained by requesting photographs from the herbarium at the University of Tokyo (TI) and the Naturalis Biodiversity Center (L). Morphological traits were observed visually and under a stereomicroscope and measured using a Mitutoyo 500-196-30 Absolute Digimatic Vernier Caliper (Kanagawa, Japan). Additionally, the original descriptions, flora, and monographs were also used as a reference.

DNA extraction and polymerase chain reaction amplification

Leaf material from each individual was collected and dried in silica gel for DNA extraction from voucher specimens (deposited in KH). Total genomic DNA was extracted from the dried leaf material using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA was electrophoresed on a 1% agarose gel to confirm the presence of DNA. The concentration and quality of the DNA were confirmed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Polymerase chain reaction (PCR) amplification of the two nrDNA regions (ITS and 5S NTS) and one cpDNA region (matK) was performed. The total volume of each PCR mix was 20 μL, comprising 15 μL of distilled water, 1.0 μL of each forward and reverse primer (50 mM), and 1 unit of Taq DNA polymerase master mix (Amplicon, Rødovre, Denmark). The primers used for PCR amplification and the PCR cycle conditions are listed in Table 1. The PCR products were visualized on 1% agarose gels and sequenced using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). The determined sequences were deposited in GenBank (Table 2). The analyzed nucleotide sequences were determined after checking the chromatogram using Geneious R 7.1.9 (Biomatters Ltd., Auckland, New Zealand). All sequences were aligned using MUSCLE software (Edgar, 2004). Polymorphic sites were identified by overlapping peaks in the chromatogram. For these polymorphic sites, we used the IUPAC ambiguity codes. We used PHASE v.2.1.1. (Stephens et al., 2001; Stephens and Scheet, 2005) to phase ambiguous sites of nuclear sequences, and format conversion was performed using the SeqPHASE online tool (Flot, 2010). The phased sequences of the samples were included in the subsequent analyses as sequence version “a” and “b” of the same samples.

Phylogenetic analysis

Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted for the two nrDNA regions (ITS and 5S NTS) and one cpDNA region (matK). For the ML analysis, the best-fit model was selected based on the Akaike information criterion implemented in the program jModelTest version 2.1.6 (Darriba et al., 2012). ML analyses were conducted using IQ-Tree 1.6.12 (Nguyen et al., 2015), applying a K3P + R2 (in ITS and 5S NTS) and TPM3u + F (in matK) substitution model. 1,000 bootstrap replicates were used to estimate the confidence values of the nodes. The BI analysis used the GTR + I substitution model determined by the Bayesian information criterion (Schwarz, 1978). The BI inference was performed using MrBayes 3.2.7 (Ronquist et al., 2012). Four Markov chain Monte Carlo chains were run for 10,000,000 generations each and sampled at every 1,000 generations, starting with a random tree. The convergence of the runs and the estimation of burn-in were checked using Tracer v.1.6 (Rambaut et al., 2014). Bayesian posterior probabilities (PP) were calculated for the majority consensus tree of all sampled trees after discarding the trees sampled within the burn-in phase (initial 25%) using MrBayes 3.2.7.

RESULTS

Morphological examination

We compared the morphological characteristics of five taxa belonging to the Elaeagnus sect. Sempervirentes. The measured values are listed in Table 3. The prominent morphological traits that were used for identification are habit, twig shape and color, presence or absence of thorns, petiole color and length, leaf shape, color of the abaxial surface of the leaf, shape of the calyx tube, hair type of style, and seed shape (Fig. 2). Based on the comparison, Elaeagnus ×maritima showed similar morphological traits to E. glabra in its scandent stems, reddish brown twigs, abaxial surface of leaves with dense reddish brown scales, gradually constricted calyx tubes, and seeds without beaks. Despite these similarities, E. ×maritima showed differences in its angled and rather thick twigs, longer petioles, broader leaves, and 4-angled and broader calyx tubes. E. ×submacrophylla showed similar morphological traits to E. pungens in its erect stem, reddish brown twig, abaxial surface of leaves with dense silvery white and sparse brown peltate scales, abruptly constricted calyx tubes, and seeds with beaks. Despite these similarities, E. ×submacrophylla showed differences in its leaf shape, calyx shape, and style with white stellate hairs.

nrDNA sequences and phylogenetic analysis

In the ITS region, the sequence length of all examined accessions was 645 bp. Excluding these gaps, the parental species showed sequence variations at eight nucleotide sites. The pairwise-sequence difference between E. glabra and E. macrophylla was five nucleotide substitutions. E. macrophylla and E. pungens differed by four nucleotide substitutions. E. glabra and E. pungens can be distinguished based on five nucleotide substitutions. However, some individuals of E. glabra (JJD178 and JJD180) and E. pungens (TY216), which grew with E. macrophylla, showed polymorphic signals for some species-specific nucleotides, making species identification difficult. Among the putative hybrid species, E. ×maritima exhibited polymorphic ITS signals at those five sites, differentiating itself from E. glabra and E. macrophylla, thus showing sequence additivity. Some individuals of E. ×submacrophylla (GJ378 and TY212) exhibited polymorphic ITS signals at four sites differentiating themselves from E. macrophylla and E. pungens, thus showing sequence additivity. The other individuals of E. ×submacrophylla showed sequences of E. macrophylla (Table 4).

In the 5S NTS, the sequence length for all examined accessions was 200 bp, except for E. glabra which had a length of 199 bp due to a 1 bp deletion at nucleotide site 60. Excluding these gaps, the putative parental species showed sequence variations at 12 nucleotide sites. The pairwise-sequence difference between E. glabra and E. macrophylla was a seven nucleotide substitutions. E. macrophylla and E. pungens differed in 11 nucleotide substitutions. E. glabra and E. pungens can be distinguished based on five nucleotide substitutions. However, similar to the ITS sequence, one individual of E. glabra (JJD178), which grew with E. macrophylla, showed polymorphic signals for some species-specific nucleotides, making species identification difficult. Among the putative hybrid species, E. ×maritima individuals exhibited polymorphic 5S NTS signals at seven sites differentiating itself from E. glabra and E. macrophylla, thus showing sequence additivity. Some individuals of E. ×submacrophylla (GJ378 and TY212) exhibited polymorphic 5S NTS signals at 10 sites differentiating themselves from E. macrophylla and E. pungens, thus showing sequence additivity. Other individuals of E. ×submacrophylla showed squences of E. macrophylla (Table 4).

The combined nrDNA sequences of the five Elaeagnus taxa contained 846 characters, comprising 796 constant, 30 parsimony-uninformative, and 20 parsimony-informative characters. In the combined nrDNA phylogenetic tree, this tree showed that it formed a polytomy. However, these five Elaeagnus taxa were divided into three clades. E. macrophylla, E. ×maritima, and E. ×submacrophylla were formed in a clade (bootstrap support [BS] = 95/PP = 1). E. pungens and E. ×submacrophylla were formed in the second clade (BS = 99/PP = 1). E. glabra and E. ×maritima were formed in the third clade (BS = 92/PP = 1). Each parental taxon was formed in a monophyletic group, and hybrid putative taxa were incorporated into the monophyletic group of each putative parental taxon (Fig. 3A).

cpDNA sequences and phylogenetic analysis

To trace the plastid donor (maternal origin) of the putative hybrid individuals, the DNA sequences of one plastid marker (matK) were examined for 24 individuals representing the putative hybrid and its closely related taxa. The size range of matK observed in the examined taxa was 1,015–1,024 bp. In the matK, sequence length for all examined accessions was 1024 bp, except E. pungens and E. ×submacrophylla which had a length of 1,015 bp due to an 8 bp deletion at nucleotide sites 1,395–1,396 and 1,437–1,442. The matK region was useful for identifying the E. pungens and E. macrophylla-E. glabra with 14 nucleotide substitutions, excluding gaps. However, this marker was unable to distinguish between E. glabra and E. macrophylla; therefore, additional markers are needed to clearly distinguish between these two taxa (Table 5).

The aligned matK sequence had a length of 1,525 characters, comprising 1,502 constant, 11 parsimony-uninformative, and 12 parsimony-informative characters. In the matK phylogenetic tree, the putative hybrid individuals and three taxa of the genus Elaeagnus were divided into two clades. E. pungens and E. ×submacrophylla were formed in one clade (BS = 100/PP = 1). E. glabra, E. macrophylla, E. ×maritima, and E. ×submacrophylla were formed in the other clade (BS = 94/PP = 1) (Fig. 3B).

DISCUSSION

This study confirmed the hybrid origins of E. ×maritima (E. glabra ×E. macrophylla) and E. ×submacrophylla (E. pungens ×E. macrophylla). For E. ×maritima, most individuals exhibited additive species-specific sites of both E. glabra and E. macrophylla in the nrDNA region. However, it was not feasible to determine the plastid donors (maternal origin), because no species-specific sites between E. glabra and E. macrophylla were observed in the cpDNA region.

E. ×submacrophylla exhibits a range of nrDNA and cpDNA characteristics. Some individuals (GJ378 and TY212) displayed sequence additivity and additive species-specific sites for both E. pungens and E. macrophylla in the nrDNA region, and E. macrophylla sequences in the cpDNA region. In contrast, other individuals (TY208 and TY211) showed an E. macrophylla sequence in the nrDNA region, with a polymorphic sequence at some species-specific nucleotides, but possessed E. pungens sequences in the cp region, revealing discrepancies between nrDNA and cpDNA. Furthermore, other individuals (GJ376 and TY213) had sequences of both E. macrophylla in the nrDNA and cpDNA regions, despite their intermediate morphology.

Hybridization can have two main effects on nrDNA sequences, resulting in either additive patterns (both parental ribotypes present) or biased homogenization toward one of the parental lineages. Although divergent ribotypes in hybrid lineages can be quickly homogenized through a process called concerted evolution (Wendel et al., 1995; Aguilar et al., 1999), some early generations of hybrid lineages can maintain the divergent ribotypes contributed by the parental species (Siripun and Schilling, 2005; Liu et al., 2009; Cho et al., 2014; Shin et al., 2014; Gil and Kim, 2016). The observed patterns of completely additive polymorphic sites and the lack of new unique mutations in E. ×maritima and E. ×submacrophylla in relation to their differentiated parental species are consistent with recent or F₁ hybrid formations (Rieseberg et al., 1993; Nieto Feliner et al., 2004). It is expected that F₂ or higher generations will possess either the nrDNA sequences of one parent only or one predominant nrDNA type, showing incomplete homogenization after a crossing-over event at a single nrDNA locus (Sang et al., 1995; Wendel et al., 1995). Therefore, intermediate situations between complete additivity and total homogenization can result in the second case.

Polyploidization and agamospermy are known to be mechanisms that can retard concerted evolution (Karvonen and Savolainen, 1993; Suh et al., 1993; Sang et al., 1995; Campbell et al., 1997). However, in Elaeagnus taxa, no cases of polyploidization or agamospermy have been reported. Since Elaeagnus taxa are shrubs, which have relatively longer generation times than herbs and subshrubs, a longer generation time may have contributed to the various aspects observed in the nrDNA region.

Three taxa, E. glabra, E. macrophylla, and E. pungens, were morphologically distinct, but there appeared to be incomplete differentiation in their development. This can be a strategic decision for evolutionary success, known as syngameon, a phenomenon observed in Fagaceae species (Cannon and Petit, 2020; Buck and Flores-Renteria, 2022).

In nature, incomplete geographic, ecological (habitat, flowering period), breeding barriers appear to have resulted in frequent hybridization between the parental taxa. First, E. glabra and E. pungens grew in mountainous areas, while E. macrophylla grew near the coast, resulting in a split ecological niche. However, in the mountains near the coast or islands, E. macrophylla grew widely to mountainous areas, and these taxa coexisted. Second, these three taxa share a flowering period from October to November, which increases the likelihood of hybridization or introgression. Finally, hybrid individuals have been observed to produce low fruit and seed yields. Nevertheless, the presence of young hybrid plants indicates that these hybrids can produce fertile seeds. In reproductive biology studies of the genus Elaeagnus, E. umbellate has variability among plants in the separation of male and female floral parts, and selffertilization through autogamy has been observed (Soley and Sipes, 2021). In order to confirm the breeding system in hybrid taxa, it seems necessary to observe floral morphology, phenology, and pollinators along with pollination experiments.

Hybridization and introgression events can complicate the inference of species phylogeny from gene trees, resulting in a polytomy (Aguilar and Feliner, 2003). Therefore, additional markers and sampling may be necessary to elucidate the relationships among these Elaeagnus species.

Key to five taxa of Elaeagnus section Sempervirentes in Korea

1. Evergreen, flowering in autumn, fruiting in spring; leaves coriaceous (sect. Sempervirentes).
- 2. Twigs and petioles grayish white or pale brown; abaxial surface midrib of leaves silvery white; style with silvery white peltate scales and white stellate hairs ···················································· E. macrophylla
- 2. Twigs and petioles reddish brown; midrib of abaxial surface of leaves reddish brown; style without silvery white peltate scales, white stellate hairs present or absent.
  - 3. Stems erect; twigs with thorns (sometimes without thorns in E. ×submacrophylla); abaxial surface of leaves with dense silvery white and sparse brown peltate scales; calyx tube abruptly constricted above the ovary; seed with beak.
    
    4. Leaves elliptic or oblong; calyx tube tubular; style without white stellate hairs ··· E. pungens
    
    4. Leaves ovate-elliptic or broadly elliptic; calyx tube campanulate; style with white stellate hairs ········ ················································· E. ×submacrophylla
  - 3. Stems scandent; twigs without thorns; abaxial surface of leaves with dense reddish brown scales or silvery white peltate scales mixed with reddish brown peltate scales; calyx tube gradually constricted above ovary; seed without beak.
    
    5. Twigs slender, smooth; petioles less than 1 cm long; leaves elliptic or narrowly elliptic; calyx tube slender, not 4-angled ················· E. glabra
    
    5. Twigs rather thick, angular; petioles more than 1 cm long; leaves ovate-elliptic, broadly elliptic, broadly ovate or orbicular; calyx tube rather broad, 4-angled ··························· E. ×maritima
1. Deciduous, flowering in spring, fruiting in autumn; leaves membranous (Sect. Elaeagnus).

Description

Elaeagnus ×maritima Koidz., Bot. Mag. (Tokyo) 31: 133, 1917.—TYPE: JAPAN. Kanagawa Pref., Sagami, Hayama, 3 Nov 1916, K.Hisautsi s.n. (lectotype, TI, seen as photo!, designated here, see Fig. 4A); Musashi, Yokohama, 5 Nov 1916, K.Hisautsi s.n. (syntype, TI, seen as photo!).

Elaeagnus ×hisauchii Makino ex Nakai, Bot. Mag. (Tokyo) 32: 223, 1918.—TYPE: KOREA. Jeju-do, 2 Nov 1917, T.Nakai 6352 (syntype, TI, seen as photo!, see Fig. 4B. JAPAN. Musashi, Yokohama, 5 Nov 1916, K.Hisautsi s.n. (syntype, TI, not seen); Yokosuka, J.Matsumura s.n. (syntype, TI, not seen).

Elaeagnus ×liukiuensis Rehder, J. Arnold Arbor. 1: 181, 1920.—TYPE: JAPAN. Okinawa (Liukiu), Okinawa Island, common round Naha, 1 Mar 1917, E.H.Wilson 8159 (holotype, A, seen as photo!; Isotype, K, US, seen as photo!).

Shrubs or vines, evergreen, about 3 m high. Stems scandent; bark shallowly or irregularly longitudinally fissured, gray or grayish brown; lenticels orbicular or elliptic, dark gray; branches gray or grayish brown, glabrous, without thorn; twigs prismatic, brown, grayish brown or gray, 1.5–2.3 mm wide, conspicuous ribs, densely reddish brown peltate scales; winter bud naked, terminal bud oblong or widely ellipsoid, 2.4–3.3 × 1.4–1.9 mm, lateral bud oblong or ovoid, 1.4–2.2 × 0.6–1.4 mm, brown to dark brown, densely reddish brown peltate scales. Leaves alternate, simple; petioles brown, grayish brown or gray, 11.3–19.2 × 1.2–2 mm, densely with reddish brown peltate scales; blades ovate-elliptic, broadly ovate, broadly elliptic or orbicular, 4.8–9.2 × 3.2–5.6 cm, apex acute to acuminate or often caudate with obtuse tip, margin entire, undulate, base obtuse to rounded, coriaceous, adaxial surface dark green, lustrous, sparsely brown peltate scales, glabrescent, abaxial surface yellowish green, densely reddish brown peltate scales or silvery white mixed with reddish brown peltate scales. Inflorescences axillary, fasciculate, 2–5 flowered; pedicels white or pale yellow, 2.6–4.6 × 0.5–0.7 mm, reddish brown peltate scales. Flowers bisexual, incomplete, apetalous, 3.9–6.2 mm in diameter; Calyx tubular, white or pale yellow, densely silvery white peltate scales mixed with reddish brown peltate scales; calyx tube rather broader, gradually constricted above ovary, 4–5.3 × 1.7–3.7 mm, 4-angled; 4-lobed, lobes triangular-ovate, 2– 3.6 × 1.7–3.5 mm, apex acute to acuminate, margin entire; stamens 4, epipetalous, antisepalous, borne in throat of calyx tube; filaments linear, white or yellowish white, ca. 0.4 mm long; anthers basifixed, narrowly oblong to elliptic, yellowish brown or brown, 1.9–2.2 × 0.8–1 mm; ovary epigynous, elliptic, 1.8–2.6 × 1–1.4 mm, 1-loculed; style linear, yellowish green or green, 4.3–5.1 mm long, glabrous or sparsely white stellate hair; stigma, clavate, bended, herkogamy absence or slightly approach hekogamy. Fruits drupe, ellipsoid, red-orange at maturity, 13.7–16.3 × 4.6–6.3 mm, silvery white peltate scales. Seeds oblong, yellowish brown, 13.3–16.1 × 4–5.8 mm, apex gradually constricted, obtuse, surface 8-grooved.

Korean name: Nok-bo-ri-ttong-na-mu (녹보리똥나무).

Phenology: Flowering October to November and fruiting April to May.

Distribution: Japan, Korea (Jeollanam-do, Gyeongsangnam-do, Jeju-do).

Habitat: Streamside, scrublands, and thickets.

Taxonomic notes: Elaeagnus ×hisauchii was similar to E. macrophylla by broadly ovate to nearly orbicular leaves and 4-angled campanulate calyx tube. However, it differed from E. macrophylla by reddish brown twigs and petioles, short petioles, small leaves, and gradually constricted calyx tube above the ovary. Therefore, it was previously treated as a synonym of E. ×submacrophylla by having the abaxial surface of leaves with dense silvery white and sparse brown peltate scales, style with white stellate hairs (Nakai, 1928; Lee, 2003). However, E. ×hisauchii in Korea was observed not only on Jeju Island, which was previously known as type locality, but also on Heuksan-do Island (see Fig. 4D), and these regions co-occur with E. glabra and E. macrophylla. Also, it differed from E. ×submacrophylla by having a vine and gradually constricted calyx tube above the ovary. Consequently, E. ×hisauchii in Korea should be considered a synonym of E. ×maritima, which is regarded as a hybrid of E. glabra and E. macrophylla.

Elaeagnus ×hisauchii was described by Nakai (1918) based on a specimen from Jeju Island (T.Nakai 6352, TI; Syntype, see Fig. 4B). However, the specimen has been identified as E. glabra. Rather, another specimen from Jeju Island (T.Nakai 6351, TI, see Fig. 4C) was identified as E. ×hisauchii and best represented the morphology mentioned in the original description. To accurately identify E. ×hisauchii, a lectotype among other syntypes should be designated. However, the lectotype could not be designated in this study because other syntypes could not be found.

Additionally, E. ×maritima was assumed as a synonym of E. glabra based on the morphological comparison (external morphology, trichome and pollen) (Ki, 2004; Koh, 2005), on the other hand, there was a view of integrating into E. ×submacrophylla (Lee, 1996). However, this species differs from E. glabra by thick and angular twigs, more than 1 cm long petioles, ovate-elliptic, broadly elliptic, broadly ovate or orbicular leaves, and rather broader and 4-angled calyx tube. In addition, it was clearly distinguished from E. ×submacrophylla by the abaxial surface of the leaves with dense reddish brown scales or silvery white peltate scales mixed with reddish brown peltate scales, gradually constricted calyx tubes, and seeds without beaks (Figs. 5, 6).

Representative specimens examined: KOREA. Jeollanam-do: Sinan-gun, Heuksan-myeon, Is. Heuksan, 31 Jan 2008, J.K. Ahn et al., SB20083 (KB); Sinan-gun, Heuksan-myeon, Ye-ri, 22 Oct 2011, B.U. Oh, Sinangun(Daeheuksando)-111022-095–096 (KH); Wando-gun, Bogil-myeon, Is. Bogildo, May 1965, L.M. Kim, KFR20015800–20015802 (KH); Wando-gun, Bogil-myeon, Yesong-ri, Suribong Peak, 7 Apr 2022, B.K. Park et al., Suribong-220407-027–028 (KH); Wando-gun, Gunoe-myeon, 25 Jun 2012, C.J. Oh et al., JN_V2012031 (KH); Wando-gun, Gunoe-myeon, Wando Arboretum, 4 Feb 2005, J.E. Koh & J.H. Kim, B200508052079, s.n. (SKK); Wando-gun, Gunoe-myeon, Daemun-ri, Wando Arboretum, 20 Jul 2013, M.S. Kang et al., 2013JNV027–028 (KH); Wando-gun, Gunoe-myeon, Yeongpung-ri, 3 Oct 2003, K.I. Heo et al. 27983, 28069, 28086, 28087, 28824 (SKK); Wando-gun, Gunoe-myeon, Yeongpung-ri, 23 Oct 2005, J.H. Lee et al. s.n. (SKK); Jindo-gun, Gogun-myeon, Jindo, 1969, L.M. Kim & M.Y. Cho, KFR20015666–20015668 (KH); Jindo-gun, Uisin-myeon, Temp. Ssanggyesa, 19 May 2002, K.I. Heo et al., 26687, 26690, 26691 (SKK); Jindo-gun, Uisinmyeon, Sacheon-ri, Mt. Cheomchalsan, Temp. Ssanggyesa, 6 Nov 2004, K.I. Heo & J.E. Koh s.n. (SKK); Jindo-gun, Uisin-myeon, Sacheon-ri, Unlimsanbang, 4 Nov 2004, K.I. Heo & J.E. Koh s.n. (SKK); Jindo-gun, Jindo-eup, Mt. Cheomchalsan, A path up a mountain of Ariranbi, 5 Nov 2004, K.I. Heo & J.E. Koh s.n. (SKK). Gyeongsangnam-do: Geoje-si, Nambu-myeon, Jeogu-ri, 22 Jun 2010, E.S. Jeon, ESJeon101305 (KH). Jeju-do: Mt. Hallasan, May 1966, L.M. Kim & S.G. Kang, KFR20015663–20015664, s.n. (KH); Jeju-si, Gujwa-eup, Songdang-ri, 21 Sep 2021, B.K. Park et al., Songdangri-210928-001 (KH); Jeju-si, Gujwaeup, Songdang-ri, 17 Oct 2021, K.H. Lee, Songdangri-211017-001–002 (KH); Jeju-si, Gujwa-eup, Songdang-ri, 23 Mar 2022, B.K. Park et al., Songdangri-220323-001–002 (KH); Gujwa-eup, Songdang-ri, 10 Oct 2022, E.S. Kang et al., Songdangri-221012-001–007 (KH); Jeju-si, Arang-dong, Yangji park, 7 Feb 2005, J.H. Kim et al. 054091, 054100, 054102, 054112 (SKK); Jeju-si, Hangyeongmyeon, Cheongsu-ri, Sanyang Keununggot, 2 Nov 2021, B.K. Park et al., Cheongsuri-211102-003–005 (KH); Jeju-si, Haean-dong (Gwangryeongcheon), 28 Sep 2012, D.S. Kim & S.Y. Kim, HALLA1701 (KH); Seogwipo-si, Sanghyo-dong, Donnaeko, 31 Oct 2005, J.H. Kim 04671, 04672, 054252 (SKK); Seogwipo-si, Sanghyo-dong, 23 Mar 2022, B.K. Park et al., Sanghyodong-220323-001–003 (KH); Seogwipo-si, Sanghyo-dong, 26 Apr 2022, Y.J. Jang & K.H. Lee, Sanghyodong-220407-007–008 (KH); Seogwipo-si, Jungmun-dong, Cheonjeyeon Waterfalls, 29 Oct 2010, C.H. Kim et al. 51828 (KB).

Elaeagnus ×submacrophylla Servett., Beih. Bot. Centralbl. 25: 84, 1909.—TYPE: JAPAN. Kyushu, Nagasaki Pref., Nomo-saki, Buerger 385 (lectotype, L, seen as photo!, designated here, see Fig. 7A); Buerger 384 (syntype, L, seen as photo!).

Elaeagnus ×nikaii Nakai, Bot. Mag. (Tokyo) 32: 224, 1918.—TYPE: JAPAN. Yamaguchi Pref., Nagato, Hagi, Tsuruedai, 3 Nov 1917, J. Nikai 2723 (lectotype, TI, seen as photo!, see Fig. 7B; isolecotype, TNS, seen as photo!).

Shrubs, evergreen, about 3 m high. Stems erect; bark shallowly or irregularly longitudinally fissured, gray or grayish brown; lenticels orbicular, dark gray; branches dark gray or grayish brown, glabrous, thorn present or absent; twigs terete, brown, grayish brown or gray, 1.6–2.2 mm wide, densely reddish brown peltate scales; winter bud naked, brown to dark brown, densely reddish brown peltate scales, terminal bud oblong or widely ellipsoid, lateral bud oblong or ovoid, 1.5–2.3 × 0.7–1 mm. Leaves alternate, simple; petioles brown, grayish brown or gray, 10.6–19.1 × 1.1–2.7 mm, densely reddish brown peltate scales; blades ovate-elliptic to broadly elliptic, 5.6–9.3 × 3.7–5.1 cm, apex acute to acuminate or often caudate with obtuse tip, margin entire, undulate, base obtuse to rounded, coriaceous, adaxial surface dark green, lustrous, sparsely silvery white peltate scales, glabrescent, reticulate vein inconspicuous when they are dry, abaxial surface yellowish green, densely silvery white peltate scales, punctured with reddish brown peltate scales. Inflorescences axillary, fasciculate, 1–3 flowered; pedicels white or pale yellow, 2.4–3.9 × 0.6–0.7 mm, reddish brown peltate scales. Flowers bisexual, incomplete, apetalous, 5.5–9.5 mm in diameter; Calyx campanulate, white or pale yellow, silvery white peltate scales and reddish brown peltate scales; calyx tube abruptly constricted above ovary, 4.9–6.2 × 2.7–4.2 mm, 4-angled; 4-lobed, lobes triangular-ovate, 2.7–4.7 × 2.2–4.4 mm, apex acute to acuminate, margin entire; stamens 4, epipetalous, antisepalous, borne in throat of calyx tube; filaments linear, white or yellowish white, 0.4–0.5 mm long; anthers basifixed, narrowly oblong to elliptic, yellowish brown or brown, 2.9–3.2 × 1–1.3 mm; ovary epigynous, elliptic, 2.2– 3.1 × 1.3–1.8 mm, 1-loculed; style linear, yellowish green or green, 8–9.3 mm long, whitish stellate hairs; stigma beyond stamens, clavate, bended, herkogamy absence or slightly approach hekogamy. Fruits drupe, ellipsoid, red-orange at maturity, 11.7–12.4 × 5.2–5.8 mm, silvery white peltate scales. Seeds oblong, yellowish brown, 10.7–11.2 × 4.2–4.8 mm, apex attenuate, beaked, surface 8-grooved.

Korean name: Keun-bo-ri-jang-na-mu (큰보리장나무).

Phenology: Flowering October to November and fruiting April to May.

Distribution: Japan, Korea (Gyeongsangnam-do).

Habitat: Streamside, scrublands, and thickets.

Taxonomic notes: Elaeagnus ×submacrophylla was described by Servettaz (1909) and is assumed to be a hybrid of E. pungens and E. macrophylla. However it was recognized as a hybrid of E. glabra and E. macrophylla by Nakai (1928), who treated E. ×hisauchii Nakai as a synonym of E. ×submacrophylla, and the hybrid of E. pungens and E. macrophylla was treated as E. ×nikaii Nakai (Lee, 1996; Lee, 2003; Ohashi, 2003). In a recent taxonomic treatment, E. ×hisauchii was treated as a synonym of E. ×maritima, which is regarded as a hybrid of E. glabra and E. macrophylla, and E. ×nikaii was treated as a synonym of E. ×submacrophylla, which is regarded as a hybrid of E. pungens and E. macrophylla (Ohashi, 2003; Ko, 2015). Elaeagnus ×submacrophylla in Japan has been known to be distributed in regions where E. pungens and E. macrophylla co-occur (Ohashi and Yoshida, 2003). But E. ×submacrophylla previously reported in Korea is confused about the distribution in that it was a region where E. glabra and E. macrophylla co-occur or only E. macrophylla occurs (Ko, 2015). As a result of the field survey and reclassification of the specimens, those identified as E. ×submacrophylla in Korea were confirmed to be either E. macrophylla or E. ×hisauchii (= E. ×maritima). In addition, E. ×nikaii in Korea was not only previously reported on Mt. Mireuksan, Tongyeong where E. pungens and E. macrophylla co-occur, but also was morphologically similar to E. macrophylla, but differed by reddish brown twigs, petioles, midrib of abaxial surface of leaves, style with no silvery white peltate scales and only white stellate hairs (Figs. 8, 9). Therefore, E. ×nikaii previously reported in Korea is considered to be E. ×submacrophylla.

Representative specimens examined: KOREA. Gyeongsangnam-do: Geoje-si, Dundeok-myeon, Sanbangri, Temp. Bohyeonsa, 30 Mar 2022, B.K. Park et al., Bohyeonsa-220330-001–002 (KH); Dundeok-myeon, Sanbang-ri, Temp. Bohyeonsa, 19 Oct 2022, Y.J. Jang et al., Bohyeonsa-221019-003–004 (KH); Tongyeong-si, Bongpyeong-dong, Mt. Mireuksan, 26 Apr 2005, J.H. Lee et al. 044754–044756, 046451–046453, 054328, s.n. (SKK); Tongyeong-si, Bongpyeong-dong, Mt. Mireuksan, 27 Oct 2021, B.K. Park et al., Mireuksan-211027-001–002 (KH); Tongyeong-si, Bongpyeong-dong, Mt. Mireuksan, 31 Mar 2022, B.K. Park et al., Mireuksan-220331-001–005 (KH); Tongyeong-si, Bongpyeong-dong, Mt. Mireuksan, 20 Oct 2022, B.K. Park et al., Mireuksan-220331-001–005 (KH); Tongyeong-si, Bongpyeong-dong, Mt. Mireuksan, 20 Oct 2022, Y.J. Jang et al., Mireuksan-220331-004 (KH).

ACKNOWLEDGMENTS

We are grateful to the persons concerned at the KB, SKK, and YNUH herbaria for permitting the examination of specimens, Dr. Akiko Shimizu (TI Herbarium) and Roxali Bijmoer (Naturalis Biodiversity Center) for providing images and allowing us to use, Hyeryun Jo for preparing the line drawings. This study was supported by the Korea National Arboretum (KNA1-1-18, 15-3).

NOTES

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

Fig. 1.

Investigated localities (TY, Tongyeong; GJ, Geoje; JJD, Jejudo Island; WD, Wando; HSD, Heuksando Island).

Fig. 2.

Illustrations of the major morphological characters among Elaeagnus section Sempervirentes taxa in Korea. A. Twig. B. Leaf (abaxial). C. Flower (longitudinally section). D. Seed. Illustrations by Hyeryun Jo.

Fig. 3.

Bayesian tree of Elaeagnus sect. Sempervirentes in Korea, (A) based on nrDNA (ITS + 5S NTS) and (B) cpDNA (matK). Numbers above branches represent bootstrap support and posterior probability (ML/BS), respectively. Putative hybrid species are indicated by black and white star shapes. nrDNA, nuclear ribosomal DNA; ITS, internal transcriped spacer; NTS, nontranscribed spacer; cpDNA, chloroplast DNA; ML, maximum likelihood; BS, bootstrap support.

Fig. 4.

Specimens of Elaeagnus ×maritima Koidz. and E. ×hisauchii Makino ex Nakai. A. Lectotype of E. ×maritima (K.Hisauchi s.n., TI, designated here). B. Syntype of E. ×hisauchii (T.Nakai 6352, TI) identified as E. glabra. C. E. ×hisauchii (Jeju-do, Korea, 3 Nov 1917, T.Nakai 6351, TI). D. E. ×maritima (Is. Heuksando, Sinan-gun, Jeollanam-do, Korea, 22 Oct 2011. B.U.Oh 111022-096, KH) previously identified as E. ×submacrophylla.

Fig. 5.

Photographs of Elaeagnus ×maritima Koidz. A. Habit. B. Bark. C. Branch. D. Twig. E. Leaf (adaxial). F. Leaf (abaxial). G. Inflorescence. H. Flower (lateral view). I. Flower (top view). J. Pistil. K. Fruit. L. Seed.

Fig. 6.

Illustrations of Elaeagnus ×maritima Koidz. A. Flowering habit. B. Fruiting habit. C. Twig. D. Winter bud. E. Leaf (adaxial). F. Leaf (abaxial). G. Leaf margin (adaxial). H. Leaf margin (abaxial). I. Petiole (adaxial). J. Leaf midrib (abaxial). K–M. Peltate scales. N. Flower (lateral view). O. Flower longitudinally section. P. Flower (top view). Q. Pistil. R. Stamen (adaxial). S. Stamen (abaxial). T. Fruit. U. Endocarp. V. Endocarp transverse section W. Seed. Illustrations by Hyeryun Jo.

Fig. 7.

Specimens of Elaeagnus ×submacrophylla Servett. and E. ×nikaii Nakai. A. Lectotype of E. ×submacrophylla (Buerger 385, L, designated here). B. Lectotype of E. ×nikaii (J.Nikai 2723, TI).

Fig. 8.

Photographs of Elaeagnus ×submacrophylla Servett. A. Habit. B. Bark. C. Branch. D. Twig. E. Leaf (adaxial). F. Leaf (abaxial). G. Inflorescence. H. Flower (lateral view). I. Flower (top view). J. Pistil. K. Fruit. L. Seed.

Fig. 9.

Illustrations of Elaeagnus ×submacrophylla Servett. A. Flowering habit. B. Fruiting habit. C. Twig (with thorn). D. Leaf (adaxial). E. Leaf (abaxial). F. Leaf margin (adaxial). G. Leaf margin (abaxial). H. Petiole (adaxial). I. Leaf midrib (abaxial). J–L. Peltate scales. M. Flower (lateral view). N. Flower longitudinally section. O. Flower (top view). P. Pistil. Q. Stamen (adaxial). R. Stamen (abaxial). S. Fruit. T. Endocarp. U. Endocarp transverse section. V. Seed. Illustrations by Hyeryun Jo.

Table 1.

List of primers and PCR conditions used in the present study.

Region	Primer name	Primer sequence (5′ – 3′)	Reference	PCR condition
ITS	ITS 5	GGA AGT AAA AGT CGT AAC AAG G	White et al. (1990)	95°C for 5 min, 35 cycles of 94°C for 20 s, 56°C for 20 s, 72°C for 1 min, 72°C for 5 min
ITS	ITS 4	TCC TCC GCT TAT TGA TAT GC	White et al. (1990)
5S NTS	5S1	GGA TGG TGA CCT CCC GGG AAG TCC	Falistocco et al. (2007)	95°C for 5 min, 35 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 30 s, 72°C for 5 min
5S NTS	5S2	CGC TTA ACT GCG GAG TTC TGA TGG G	Falistocco et al. (2007)
matK	trnK685F	GTA TCG CAC TAT GTA TCA TTT GA	Wojciechowski et al. (2004)	95°C for 5 min, 35 cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 1 min, 72°C for 5 min
	matK832R	TTG CAT AGA AAT AGA TTC GCT CAA A
	matK4La	CCT TCG ATA CTG GGT GAA AGA T
	matK1932Ra	CCA GAC CGG CTT ACT AAT GGG
	matK1100L	TTC AGT GGT ACG GAG TCA AAT G
	trnK2R	CCC GGA ACT AGT CGG ATG G

PCR, polymerase chain reaction; ITS, internal transcribed spacer; NTS, non-transcribed spacer.

Table 2.

List of taxa used for nrITS and cpDNA sequences analysis with voucher and GenBank accession numbers.

Taxon	Locality and voucher specimen	nrDNA		cpDNA

		ITS	5S NTS	matK
Elaeagnus glabra	Myosanbong Peak, Gujwa-eup, Jeju-si, Jeju-do, Korea Myosanbong-220323-001 (JJD178)	OQ718935	OQ714343	OQ714367
	Sanghyo-dong, Seogwipo-si, Jeju-do, Korea Sanghyodong-220323-007 (JJD180)	OQ718936	OQ714344	OQ714368
	Wando Arboretum, Daemun-ri, Wando-gun, Jeollanam-do, Korea 2013JNV027 (WD257)	OQ718937	OQ714345	OQ714379
	Wando-eup, Wando-gun, Jeollanam-do, Korea S-0568 (WD299)	OQ718939	OQ714347	OQ714381
	Ye-ri, Heuksan-myeon, Sinan-gun, Jeollanam-do, Korea Sinangun(Daeheuksando)-171022-111 (HSD298)	OQ718938	OQ714346	OQ714380
E. macrophylla	Bomok, Bomok-dong, Seogwipo-si, Jeju-do, Korea Bomok-220323-001 (JJD182)	OQ718944	OQ714352	OQ714369
	Temp. Bohyeonsa, Sanbang-ri, Geoje-si, Gyeongsangnam-do, Korea Bohyeonsa-220330-001 (GJ207)	OQ718949	OQ718949	OQ714370
	Temp. Milaesa, Tongyeong-si, Gyeongsangnam-do, Korea Milaesa-220331-001 (TY218)	OQ718945	OQ714353	OQ714371
	Gugyedeung, Wando-gun, Jeollanam-do, Korea Gugyedeung-220406-001 (WD222)	OQ718946	OQ714354	OQ714372
	Ye-ri, Heuksan-myeon, Sinan-gun, Jeollanam-do, Korea Heuksando(Yeri)-171009-012 (HSD305)	OQ718947	OQ714355	OQ714382
E. pungens	Hogok437, Suryeok-ri, Geoje-si, Gyeongsangnam-do, Korea Suryeokri-221019-001 (GJ202)	OQ718950	OQ718950	OQ714386
	Dundeok Fortress, Georim-ri, Geoje-si, Gyeongsangnam-do, Korea Dundeok Fortress-220330-001 (GJ205)	OQ718951	OQ718951	OQ714387
	Mt. Mireuksan, Bongpyeong-dong, Tongyeong-si, Gyeongsangnam-do, Korea Mireuksan-220331-011 (TY216)	OQ718952	OQ714360	OQ714388
E. ×maritima	Songdang-ri, Gujwa-eup, Jeju-si, Jeju-do, Korea Songdangri-220323-001 (JJD198)	OQ718948	OQ714356	OQ714373
	Sanghyo-dong, Seogwipo-si, Jeju-do, Korea Sanghyodong-220426-007 (JJD243)	OQ718941	OQ714349	OQ714374
	Gwangnyeongcheon River, Haean-dong, Jeju-si, Jeju-do, Korea HALLA1701 (JJD255)	OQ718942	OQ714350	OQ714375
	Suribong Peak, Bogil-myeon, Wando-gun, Jeollanam-do, Korea Suribong-220407-027 (WD233)	OQ718940	OQ714348	OQ714383
	Ye-ri, Heuksan-myeon, Sinan-gun, Jeollanam-do, Korea Sinangun(Heuksando)-111022-095 (HSD252)	OQ718943	OQ714351	OQ714385
E. ×submacrophaylla	Temp. Bohyeonsa, Sanbang-ri, Geoje-si, Gyeongsangnam-do, Korea Bohyeonsa-221019-003 (GJ376)	OQ718955	OQ714363	OQ714384
	Temp. Bohyeonsa, Sanbang-ri, Geoje-si, Gyeongsangnam-do, Korea Bohyeonsa-221019-004 (GJ378)	OQ718958	OQ714366	OQ714376
	Mt. Mireuksan, Bongpyeong-dong, Tongyeong-si, Gyeongsangnam-do, Korea Mireuksan-220331-001 (TY208)	OQ718953	OQ714361	OQ714389
	Mt. Mireuksan, Bongpyeong-dong, Tongyeong-si, Gyeongsangnam-do, Korea Mireuksan-220331-002 (TY211)	OQ718956	OQ714364	OQ714390
	Mt. Mireuksan, Bongpyeong-dong, Tongyeong-si, Gyeongsangnam-do, Korea Mireuksan-220331-003 (TY212)	OQ718957	OQ714365	OQ714377
	Mt. Mireuksan, Bongpyeong-dong, Tongyeong-si, Gyeongsangnam-do, Korea Mireuksan-220331-004 (TY213)	OQ718954	OQ714362	OQ714378

nrITS, nuclear ribosomal internal transcribed spacer; cpDNA, chloroplast DNA; NTS, non-transcribed spacer.

Table 3.

Comparison of the morphological characters among Elaeagnus section Sempervirentes taxa in Korea.

	E. glabra	E. ×maritima	E. macrophylla	E. ×submacrophylla	E. pungens
Stem	Scandent	Scandent	Scandent	Erect	Erect
Thorn	Absent	Absent	Absent	Present or absent	Present
Twig	Slender, smooth, reddish brown	Rather thick, angular, reddish brown	Rather thick, angular, grayish white or pale brown	Rather thick, angular, reddish brown	Slender, smooth, reddish brown
Petiole	Reddish brown	Reddish brown	Grayish white or pale brown	Reddish brown	Reddish brown
Length of petiole (cm)	0.7–1.3	1.1–1.7	1.2–2.3	0.9–1.5	0.7–1.4
Leaf shape	Narrowly elliptic to elliptic	Ovate-elliptic, elliptic, broadly elliptic or orbicular	Broadly ovate, oval or nearly orbicular	Ovate-elliptic to broadly elliptic	Elliptic or oblong
Leaf size (cm)	3.8–8.2 × 1.8–4.3	4.2–9.2 × 3.2–5.6	5–10.8 × 2.9–8.5	5.8–13.9 × 3.2–9	4.3–9.2 × 1.7–4.1
Abaxial surface of leaf	Densely reddish brown peltate scales or reddish brown mixed with silvery white peltate scales	Densely reddish brown peltate scales or silvery white mixed with reddish brown peltate scales	Densely silvery white peltate scales, mixed with brown peltate scales, glabrescent	Silvery white peltate scales, punctured with reddish brown peltate scales	Densely silvery white peltate scales, punctured with reddish brown peltate scales.
Calyx shape	Funnelform-tubular, gradually constricted	Tubular, gradually constricted	Campanulate, abruptly constricted	Campanulate, abruptly constricted	Tubular, abruptly constricted
Calyx size (mm)	4.1–6.1 × 1.5–2.4	3.7–5.6 × 2–3.4	4.8–6.4 × 3.4–3.8	5.5–6.2 × 3.5–4.1	4.8–8.6 × 2.1–4.1
Style	Glabrous or rarely white stellate hairs	Glabrous or white stellate hairs	Silvery white peltate scales and white stellate hairs	White stellate hairs	Glabrous
Achene	Apex gradually constricted, obtuse	Apex gradually constricted, obtuse	Apex gradually constricted, obtuse	Apex attenuate, beaked	Apex attenuate, beaked or obtuse

Table 4.

Nuclear species-specific sites in Elaeagnus section Sempervirentes taxa in Korea.

Taxon	Voucher	Locality	ITS								5S NTS

			112	125	192	466 504		532	630	631	60	62	66	72	76	87	88	111	114	144	157	191
E. glabra	JJD178	Jeju	G/C	T	T/C	T	T	C	A	T/G	-	A	A	C	A/C	A/G	T	T/C	C	C/G	A/C	A
	JJD180	Jeju	A/C	T/C	T	T/C	T	C	A	G	-	A	A	C	C	A	T	T	C	C	A/C	A
	WD257	Wando	G	T	T	T	T	C	A	G	-	A	A	C	C	A	T	T	C	C	A/C	A
	WD299	Wando	A/G	T	T/C	T	T	C	A	G	-	A	A	C	C	A	T	T	C	C	A/C	A
	HSD298	Huksando	A/G	T	T/C	T	T	C	A	G	-	A	A	C	C	A	T	T	C	C	A/C	A
E. macrophylla	JJD182	Jeju	C	C	C	C	T	C	A/C	T/G	A/T	C	A	C	A	G	T	C	C	G	C	G/A
	TY218	Tongyeong	C	C	C	C	T	C	A/C	T/G	A/T	C	A	C	A	G	T	C	C	G	C	G/A
	WD222	Wando	C	C	C	C	T	C	A/C	T/G	A/T	C	A	C	A	G	T	C	C	G	C	G/A
	HSD305	Huksando	C	C	C	C	T	C	A/C	T/G	A/T	C	A	C	A	G	T	C	C	G	C	G/A
E. pungens	GJ202	Geoje	C	C	C	C	C	A	C	G	T	A	T	G	C	A	C/T	T	G	C	C	A
	GJ205	Geoje	C	C	C	C	C	A/C	C	G	T	A	T	G	C	A	C/T	T	G	C	C	A
	TY216	Tongyeong	C	C	C	C	C	A/C	C	T/G	T	A	A/T	G/C	C	A	C/T	T	G	C	C	A
E. ×maritima	JJD198	Jeju	G/C	T/C	T/C	T/C	T	C	A	T/G	A/T	A/C	A	C	A/C	A/G	T	T/C	C	C/G	A/C	G/A
	JJD243	Jeju	G/C	T/C	T/C	T/C	T	C	A/C	T/G	-	A/C	A	C	A/C	A/G	T	T/C	C	C/G	A/C	A
	JJD255	Jeju	G/C	T/C	T/C	T/C	T	C	A	T/G	-	A/C	A	C	A/C	A/G	T	T/C	C	C/G	A/C	A
	WD233	Wando	C	T/C	T/C	T/C	T	C	A/C	T/G	-	A/C	A	C	A/C	A/G	T	T/C	C	C/G	A/C	A
	HSD252	Huksando	A/C	T/C	C	T/C	T	C	A/C	G	-	A/C	A	C	A/C	A/G	T	T/C	C	C/G	A/C	A
E. ×submacrophaylla	GJ376	Geoje	C	C	C	C	T/C	A/C	C	G	T	C	A	C	A	G	T	C	C	G	C	G/A
	GJ378	Geoje	C	C	C	C	T/C	A/C	A/C	T/G	T	A/C	A/T	G/C	A/C	G/A	C/T	T/C	C/G	G/C	C	G/A
	TY208	Tongyeong	C	C	C	C	T/C	A/C	A/C	T/G	T	a/C	A	C	A	G	T	C	C	G	C	G/A
	TY211	Tongyeong	C	C	C	C	T	C	A/C	T/G	T	C	A	C	A	G	T	C	C	G	C	G/A
	TY212	Tongyeong	C	C	C	C	T/C	A/C	A/C	T/G	T	A/C	A/T	G/C	A/C	G/A	C/T	T/C	C/G	G/C	C	G/A
	TY213	Tongyeong	C	C	C	C	T/C	A/C	C	G	T	C	A	C	A	G	T	C	C	G	C	G/A

Gray shades indicate distinct bases or gaps segregating each taxon from the other two.“-” denotes a gap.

ITS, internal transcribed spacer; NTS, non-transcribed spacer.

Table 5.

Plastid species-specific sites in Elaeagnus section Sempervirentes taxa in Korea.

Taxon	Voucher	Locality	matK

			40	73	75	156	237	704	790	1020	1022	1033	1074	1230	1395–1397	1437–1442	1489	1507
E. glabra	SOK-178	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-180	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-257	Wando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-299	Wando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-298	Huksando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
E. macrophylla	SOK-182	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-218	Tongyeong	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-222	Wando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	SOK-305	Huksando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
E. pungens	SOK-202	Geoje	A	C	A	C	T	T	A	G	C	A	A	A	---	------	A	C
	SOK-205	Geoje	A	C	A	C	T	T	A	G	C	A	A	A	---	------	A	C
	SOK-216	Tongyeong	A	C	A	C	T	T	A	G	C	A	A	A	---	------	A	C
E. ×maritima	JJD198	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	JJD243	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	JJD255	Jeju	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	WD233	Wando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	HSD252	Huksando	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
E. ×submacrophaylla	GJ376	Geoje	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	GJ378	Geoje	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	TY208	Tongyeong	A	C	A	C	T	T	A	G	C	A	A	A	---	------	A	C
	TY211	Tongyeong	A	C	A	C	T	T	A	G	C	A	A	A	---	------	A	C
	TY212	Tongyeong	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G
	TY213	Tongyeong	C	A	G	T	A	C	G	C	C	A	T	G	AGA	TTCTAC	C	G

Gray shades indicate distinct bases or gaps segregating each taxon from the other two.“-” denotes a gap.

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