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Korean J. Pl. Taxon > Volume 53(4); 2023 > Article
/home/virtual/kjpt/journal//../xmls/kjpt-53-4-264.xml LEE, CHOI, EUO, KIM, JANG, SO, SON, GANTSETSEG, JEONG, HAN, and LEE: Evergreen forests on the Korean Peninsula faced a new threat: Schizomyia castanopsisae (Jat-bam-na-mu-hok-pa-ri) inducing galls on reproductive organs of Castanopsis sieboldii in the Yokjido Island Natural Monument Forest

Abstract

Unnoticed and silently, a certain midge has emerged that could pose a threat to the evergreen forests on the Korean Peninsula and the associated island ecosystems. We report the discovery of an unrecorded species on the Korean Peninsula, identified as Schizomyia castanopsisae Elsayed & Tokuda, 2018 (Diptera: Cecidomyiidae). This previously unrecorded gall midge species induces galls on a reproductive organ of Castanopsis sieboldii, specifically the inflorescences, and was found in the natural monument forest on Yokjido Island. A total of 40 larvae were collected from 8 inflorescence galls, each obtained from individual hosts within the forest. Morphological examinations and mitochondrial DNA cytochrome oxidase subunit I (COI) region sequencing were conducted for each specimen, confirming the identification of the gall inducer as S. castanopsisae. The COI sequences from the 40 specimens collected on Yokjido Island were identical, showing a 99.8% match with individuals from Kyushu, Japan. Additionally, a maximum likelihood tree analysis revealed a sister relationship between Yokjido Island and Kyushu individuals. Considering the tropical nature of gall midge species, it suggests recent northward migration from Kyushu to Yokjido Island. This species not only significantly reduces acorn production but also has the potential to negatively impact organisms associated with acorns. Drawing from cases in Japan, this species has the potential to rapidly dominate evergreen broad-leaved forests on numerous islands in the Korean Peninsula. Therefore, urgent additional research on aspects such as natural enemies of this species, its dispersal mechanisms, and other relevant factors is necessary. We also investigated the distribution of trees with inflorescences gall-induced by S. castanopsisae in the Yokjido Island Natural Monument Forest. The occurrence frequency of these galls is estimated to be approximately 10 percent.

INTRODUCTION

Fagaceae, especially the evergreen broad-leaved trees, plays an ecologically important role as the primary component species in the evergreen forests of both the Jejudo Floristic Province and the Southern Coast Floristic Province on the Korean Peninsula. These evergreen forests are also associated with island ecosystems, making them a subject of considerable interest in terms of evolutionary, taxonomic, and conservation perspectives to many researchers (Han et al., 2020, 2023; Jang et al., 2023a). In this context, while surveying a natural monument C. cuspidata forest to elucidate the taxonomic identity of C. cuspidata (Thunb.) Schottky on the Korean Peninsula (see Jang et al., 2023b), we discovered galls on C. sieboldii (Makino) Hatus. ex T. Yamaz. & Mashiba, presumably caused by an unknown parasitic species. These parasitic species require special attention because they can have a negative impact on the ecosystem. The issues caused by parasitic species are clearly illustrated through the precedent of the Castanea species. Cryphonectria parasitica (Murr.) Barr., originated from East Asia, has caused a severe decline in native chestnut species (C. dentata (Marshall) Borkh., C. sativa Mill.) in the United States and Europe, continuing to exert negative impacts on nut production (Rigling and Prospero, 2018). Therefore, we believe that identifying and reporting the identity of the parasitic species is the first step in formulating conservation policies that can minimize damage to evergreen broad-leaf forests on the Korean Peninsula.
Schizomyia castanopsisae Elsayed & Tokuda, 2018 (Diptera: Cecidomyiidae) targets the reproductive organs of Castanopsis tree species for reproduction (Elsayed et al., 2018), in contrast to previously reported Schizomyia species in Korea, which target vegetative organs. For example, S. humuli (Shinji, 1939) and S. sasakii (Monzen, 1937) induce galls on the leaves and axillary buds of their host plants (Humulus japonicus Siebold & Zucc. and Ilex crenata Thunb., respectively). Their selection, inducing the inflorescence galls, can pose a threat to biodiversity and ecosystem services. This not only causes severe losses in acorn production, but also affects acorn-associated organisms such as birds and rodents (Wolff, 1996; Tokuda et al., 2022). S. castanopsisae is currently considered to be undergoing a rapid expansion of its distribution into higher latitude regions in Japan (Tokuda et al., 2023). The occurrence of inflorescence galls was initially found in 1980 on Okinawa (Yamauchi et al., 1982) and has progressively extended to the southern region of Kyushu, Miyazaki, as of 2010 (Tokuda et al., 2019). Notably, according to the most recent study (2022), its range has expanded further northward to the Izu Peninsula on Honshu (Tokuda et al., 2023).
Here, we present a new distribution record of the gall midge S. castanopsisae on Yokjido Island on the Korean Peninsula, which is located at a higher latitude than Fukuoka, Japan. This marks the first documented occurrence of this species on the Korean Peninsula. Its host, C. sieboldii, is a major component of evergreen broad-leaved forests on numerous islands in the Korean Peninsula. Island ecosystems are vulnerable to invasive species and other catastrophic events related to human activities (Reaser et al., 2007; Keppel et al., 2014; Tokuda et al., 2022). This is even more important considering, the abundance of islands on the Korean Peninsula, where evergreen broad-leaved forests may be at risk of the sudden occupation by the gall midge. To identify the gall-inducing insect, we examined the larval morphology of the gall midges found on Yokjido Island and subsequently confirmed the gall inducer by comparing it with DNA sequences of S. castanopsisae, previously documented by Elsayed et al. (2018). Additionally, we investigated the distribution of trees having inflorescences galled by S. castanopsisae on the Yokjido Island Natural Monument Forest.

MATERIALS AND METHODS

Galls collection and gall occurrence survey

We collected 8 inflorescence galls induced by S. castanopsisae in a natural monument of Yokjido Island, each from individual C. sieboldii trees on 2 November 2023. Following their collection, the galls were carefully placed into ziplock bags and transported to the Plant Systematic Laboratory of the Department of Biological Education at Chonnam National University. Subsequently, they were stored in a –20°C freezer. The larvae obtained from collected galls were utilized for DNA extraction, totaling 40 individuals. To estimate the frequency of gall occurrence on C. sieboldii individuals within the population, we surveyed both the total number of individuals and the specific count of individuals with gall formations. This survey targeted individuals with a breast height diameter of 20 cm or more, aiming to cover the entire population of C. sieboldii within the natural monument forest of Yokjido Island. The occurrence of galls was checked by visual inspection. The locations were recorded using GPSMAP 64st equipment (Garmin, Olathe, KS, USA) to ensure accuracy.

Morphological and molecular analyses

The morphological identification was carried out following methods described by Elsayed et al. (2018). Consecutive digital images on different focal planes (usually 50 or more shots per specimen) were obtained with a Dhyana 400dc camera (Tucsen Photonics, Fuzhou, China) mounted on a Leica M205C stereomicroscope (Leica Microsystems, Wetzlar, Hesse, Germany). Genitalia photographs were taken with a Dhyana 400dc camera (Tucsen Photonics) mounted on a Leica DM3000 LED microscope (Leica Microsystems). The images were stacked using Helicon Focus software (version 7.6.6, Helicon Soft, Ltd., Kharkiv, Ukraine). For visualization, we edited the images using Adobe Photoshop (version 22.5, Adobe Systems Inc., San Jose, CA, USA).
Total genomic DNA was extracted from the legs of a specimen using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. In this case, 40 specimens were sequenced for a 658 bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene. The DNA barcode was amplified using the primers LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al., 1994). PCR conditions for amplification followed the manufacturer’s protocol (Platinum Taq, Invitrogen, Carlsbad, CA, USA). The amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) and were directly sequenced at Macrogen (Seoul, Korea). The sequence identified in this study was deposited into GenBank (OR841482–OR841511, OR841513–OR841522).
For a phylogenetic analysis, we used 40 well-sequenced COI sequences collected from Yokjido Island. Additionally, we downloaded the COI sequences of 45 individuals of S. castanopsisae, with S. achyranthesae and S. asteris as an outgroup, from publicly available data on NCBI (https://www.ncbi.nlm.nih.gov/, accessed Nov 2023) (Table 1). The sequences were examined, edited, and aligned using Geneious Prime 22.2.1 (Kearse et al., 2012). A maximum likelihood analysis was constructed using the W-IQ-TREE Server (http://iqtree.cibiv.univie.ac.at/) (Trifinopoulos et al., 2016). Each aligned sequence dataset was tested to determine the best-fit model by using W-IQ-TREE with the Akaike criterion, and new model selection procedure. GTR+F+I was confirmed as the best-fit model. We evaluated the node supported by 1,000 ultrafast bootstrap replicates (Minh et al., 2013).

RESULTS AND DISCUSSION

We identified the gall midge collected from C. sieboldii in the Yokjido Island Natural Monument Forest as S. castanopsisae. This marks the first documentation of S. castanopsisae on the Korean Peninsula. We propose the Korean name, “Jat-bam-na-mu-hok-pa-ri” for this species. The larval morphology of gall midges is in good agreement with the original description of S. castanopsisae according to the following larval characteristics: a sternal spatula with the posterior portion about 2.8 times as wide as the base of the anterior free portion, and the anus opening branched (Fig. 1B, C) (Figs. 39, 40 in Elsayed et al., 2018). Similar to the morphological observations, the molecular analysis also showed that the mitochondrial COI region’s partial sequences (421 bp) from the gall midges collected on Yokjido Island were most similar to S. castanopsisae compared to other Schizomyia species. All 40 samples collected from Yokjido Island exhibited identical COI sequences, with sequence differences ranging from 98.9% (Nansei Islands) to 99.8% (Kyushu) when compared to other regions.
We believe that species migrated to Yokjido Island from its more southern region, given its tropical nature. It is likely that S. castanopsisae on Yokjido Island originates from the Kyushu region. This assumption is supported by the phylogenetic tree, with individuals from Yokjido Island closely related to those from Kyushu, demonstrating robust (94%) bootstrap support and forming a sister relationship with the Kyushu individuals (Fig. 2). However, the timing of this species’ initial migration from Kyushu to Yokjido Island remains unclear. We propose that the species recently migrated. Our results indicated that out of a total of 230 individuals, 20 trees on Yokjido Island exhibited inflorescence galls induced by S. castanopsisae (Fig. 3A–C). Furthermore, the COI nucleotide sequences of all surveyed individuals of S. castanopsisae on Yokjido Island were identical. Although determining the precise timing of an insect invasion event is challenging, the absence of polymorphism of COI and the low gall occurrence rate (approximately 10%) on C. sieboldii individuals in the Yokjido Natural Monument forest suggest a recent S. castanopsisae invasion, likely within the last decade. On another front, there was a reliable statement from a resident of Yokjido Island that the nut harvest of C. sieboldii has decreased since 2022 (Fig. 3B). As this was not a formal interview but rather an individual’s opinion, pinpointing the precise timing of their initial migration to Yokjido Island from such a statement is also challenging.
In Japan, there is a concern that this species will have a detrimental impact on the ecosystem by significantly reducing the production of acorns. For example, in the Izu Islands, it has been reported that nut production of C. sieboldii has decreased over the last few decades due to the high density of galls (Tokuda et al., 2019). S. castanopsisae on the Korean Peninsula may undergo stages similar to those observed in Japan. Therefore, additional research on aspects such as natural enemies of this species, its dispersal mechanisms, and other relevant factors is urgent.

ACKNOWLEDGMENTS

This study was supported by the Korea National Arboretum (KNA1-1-18, 15-3; KNA1-1-12, 16-1).

NOTES

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.

Fig. 1.
Photographs of Schizomyia castanopsisae larva. A. Body (ventral). B. Spatula. C. Anus.
kjpt-53-4-264f1.jpg
Fig. 2.
Distribution map of Schizomyia castanopsisae and maximum likelihood tree based on the cytochrome oxidase subunit I (COI) region of S. castanopsisae with S. achyranthesae and S. asteris as an outgroup (circle: haplogroup linked by common ancestor).
kjpt-53-4-264f2.jpg
Fig. 3.
A. Distribution map of Castanopsis sieboldii in the Yokjido Island Natural Monument Forest, showing individuals with galls (red) and without galls (white). B. Non-galled infructescence and nuts (collected by a resident of Yokjido Island) of C. sieboldii. C. Infructescence galled by Schizomyia castanopsisae and cross-section of a gall.
kjpt-53-4-264f3.jpg
Table 1.
Information on Schizomyia castanopsisae occurrences and GenBank accession numbers.
Country Locality First collection date GenBank acc. No. Collector
Korea Gyeongsangnam-do
  Yokjido Island 3 Nov 2023 OR841482–OR841511, OR841513–OR841522 Y.J. Jang & E.D. Lee
Japan Kyushu
  Fukuoka 19 Nov 2018 LC480920 Tokuda et al.i
  Miyazaki 2010 - Nagaib
  Kagoshima 1 Nov 2012 LC480919 Tokuda et al.i
Izu Peninsula, Shizuoka
  Irouzaki, Minamiizuchou 29 Nov 2022 LC754252–LC754253 Tokuda et al.k
Izu Islands
  Ohshima 2016 - Tokuda et al.k
  Toshima 27 Nov 2022 - Tokuda et al.k
  Niijima 11 Dec 2014 - Tokuda et al.g
  Shikinejima 15 May 2013 - Tokuda et al.e
10 Dec 2014 LC708181–LC708184 Tokuda et al.j
  Kouzushima 14 Dec 2011 LC708185–LC708186 Tokuda et al.f, j
  Miyakejima 15 Nov 2011 - Tokuda et al.c
20 Dec 2011 LC708187–LC708188 Tokuda et al. j
  Mikurajima 17 Nov 2011 LC708189–LC708192 Tokuda et al.c
  Hachijojima 21 Sep 2011 Tokuda et al.c
6 Dec 2014 LC426404–LC426406 Elsayed et al.h
  Aogashima 20 Nov 2012 LC708193–LC708195 Tokuda et al. j
Nansei Islands
  Kunigami, Okinawa 24 Oct 2008 - Ogata et al.d
Nov 2020 LC708196–LC708198 Tokuda et al. j
2 Oct 2022 LC754262–LC754265 Tokuda et al.k
  Naze, Amami-Oshima Aug & Oct 2022 LC754257–LC754260 Tokuda et al.k
  Setouchi, Amami-Oshima Aug & Oct 2022 LC754254–LC754256 Tokuda et al.k
  Nishiakina, Tokunoshima 20 Oct 2022 LC754261 Tokuda et al.k
  Okinawa 1980 - Yamauchi et al.a
  Nago, Okinawajima Oct 2022 LC754266–LC754269 Tokuda et al.k
  Higashi, Okinawajima Oct 2022 LC754270–LC754273 Tokuda et al.k

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