﻿An updated checklist of Collembola in Taiwan, with DNA barcoding of Papirioidesjacobsoni Folsom, 1924 (Symphypleona, Dicyrtomidae)

﻿Abstract From urban green space to pristine forest, Collembola is one of the most numerous and species-rich members of the soil fauna around the world. However, due to lack of taxonomic expertise and research, its diversity is poorly understood, especially in tropical and subtropical regions. Collembola biodiversity studies in Taiwan have not seen much progress since 1981, when Hsin Chi reviewed 26 species belonging to 20 genera and eight families. Additionally, reports of new records in Taiwan in the last 40 years are scattered amongst several publications and not easily accessible to most end-users. Thus, a concise summary of related research is urgently needed. In this study, we updated the checklist of Collembola in Taiwan, based on published papers as well as images recorded in 2020–2022. We concluded that 58 species of Collembola belonging to 31 genera and 12 families have been reported in Taiwan, including 13 newly-recorded species. This species richness marks a 123% increase from the 1981 review. The results have been made publicly available in the Catalog of Life in Taiwan database and the images recorded have been used to update species information in collembola.org. We also characterised morphological and genetic variations in the globular springtail species Papirioidesjacobsoni Folsom, 1924 using DNA barcodes and highlighted potential research directions.


Introduction
Springtails are microarthropods in the class Collembola (Arthropoda: Hexapoda). They are commonly found in leaf litter and soil and on the surface of plants, fungal sporocarp, decaying wood and rocks. They are one of the most abundant animals in the litter-soil habitat, with a density of up to 40,000 individuals per square metre in the soil in temperate grasslands or forests (Orgiazzi et al. 2016). Their morphologies are characterised by ventral tube/collophore on the first abdominal segment, which helps anchor themselves to the surface, as well as furca/furcula, the structure allowing them to jump. In some taxonomic groups, this latter structure is reduced and, therefore, species in these groups lost the ability to jump. Globally, about 9,000 species of Collembola have been described so far and the estimated number of species is about 50,000 to 65,000 (Bellinger et al. 1996(Bellinger et al. -2022. Most of our knowledge about this diversity comes from studies conducted in the temperate region, whereas the subtropical region has received little attention (Potapov et al. 2020).
Taiwan is an East Asian Island located between Japan and the Philippines. It has a land area of about 32,260 km 2 and is divided by the Tropic of Cancer into a humid subtropical climate in the north and a tropical monsoon climate in the south, with a mean annual precipitation of approximately 2,600 mm, mostly in the form of rainfall. The terrain on the Island was shaped by the collision between the Eurasian Plate and the Philippine Sea Plate in the last five million years (Huang et al. 1997(Huang et al. , 2000. Geographically, it is divided into the flat to gently rolling plains in the west and the rugged, forest-covered mountains in the eastern two-thirds of the Island, with over 100 mountain peaks exceeding 3,000 m in elevation. Some of these summits were covered by glaciers during the last glaciation (Ono et al. 2005) and are still regularly receiving snow and short periods of ice cover during winter nowadays. The complex terrain, climate and geological history of Taiwan, presumably, provide ample opportunity for the diversification of Collembola, as well as varying vegetation and habitats for these organisms to thrive.
Following "An Index to the Collembola" for scientific names (Salmon 1964), the first and by far the only review of Collembola in Taiwan was a Chinese-written article by Hsin Chi in 1981, which listed 26 species belonging to 20 genera and eight families (Chi 1981). As Chi noted, studies of Collembola in Taiwan during the early years were mainly conducted by Japanese researchers. The first publication was by J.R. Denis (1929), which reported three species collected from Taipei by F. Silvestri. After that, Japanese taxonomists R. Yosii and H. Uchida reported several species of Collembola in Japan and neighbouring countries and up to 37 species from Taiwan were included (Yosii 1940(Yosii , 1963(Yosii , 1965(Yosii , 1977Uchida 1943Uchida , 1955Uchida , 1956Uchida , 1957aUchida , 1957bUchida , 1958aUchida , 1958bUchida , 1959aUchida , 1959bUchida , 1960. As some of the records were later considered synonyms, the total number of species reported during this period was higher than that in the checklist compiled by Chi (1981). In addition, some Taiwanese species were occasionally recorded in entomological literature (Shiraki 1932(Shiraki , 1954Asahina et al. 1965) and an article about sugar cane pests (Takano and Yanagihara 1939).
In addition to Chi's (1981) comprehensive checklist, another 22 species have been reported in Taiwan by researchers from China, Korea and Japan. Lee and Park (1989) reported 11 species and seven genera in family Entomobryidae, including four new species and three new records. A year later, Lee and Kim (1990) reported five new species and two new records in family Neanuridae. In 2010, a subspecies of Homidia (Entomobryidae) was re-described and elevated to species level (Shi et al. 2010). Moreover, several new records were sporadically reported (Yosii 1966(Yosii , 1982Zhao et al. 1997). In contrast, studies conducted by Taiwanese researchers were mainly about pest control or survey of ground or soil arthropods, which only recorded the total number of individuals of Collembola without any detailed taxonomic information (Chen et al. 2020).
Taken together, our knowledge on the diversity of Taiwanese collembolan fauna has changed considerably in the last 40 years since Chi's comprehensive review, including changes in scientific names and synonyms. In this study, we updated the checklist of Collembola in Taiwan, based on published papers as well as images we recorded in 2020-2022. During our field sampling, we noticed apparent variations in the colour pattern of the species Papirioides jacobsoni Folsom, 1924, calling into question whether the different colour morphs are, indeed, the same species. Thus, we hypothesised that these colour morphs represent two different species and conducted DNA barcode analysis to test this hypothesis.

Materials and methods
The revised checklist is based on both published studies and newly-collected samples. Most of the sampling sites are hiking trails in forests and urban areas in northern Taiwan, with only a few samples from eastern and central Taiwan. Collembola were collected using one of the two methods; (1) Litter and surface soil were collected and then transported to the laboratory within 24 hours. Collembola were extracted from litter and soil using a Berlese-Tullgren funnel for about 5-7 days. Specimens were extracted into either a jar containing 85% ethanol or a container filled with the mixture of Plaster of Paris and fine powder of activated charcoal (Plaster of Paris: activated carbon: water = 9:1:11.25); (2) For specimens that were directly spotted in the field, an aspirator was used to collect them. The collected specimens were either kept alive for as long as possible in a container filled with the mixture of Plaster of Paris and activated charcoal or stored in 85% ethanol at 4 °C for future molecular study.
Live and ethanol-preserved specimens were examined under a Nikon SMZ800N stereomicroscope, equipped with a plan Apo 1× objective lens to reduce chromatic aberration and a TOUPCAM E3ISPM12300KPA digital camera for photography. Species identification is based on Bretfeld (1999), Potapov (2001) andJordana (2012). For families, scientific names and synonyms, we followed the Checklist of the Collembola of the World maintained by Bellinger et al. (1996Bellinger et al. ( -2022 and hosted in collembola. org. In most cases, junior synonyms were listed when they were related to previous records of Taiwanese Collembola. Whenever available, additional information about locations and habitats of a species was detailed in the Remarks. Species marked with an asterisk (*) are new records identified based on photographs of live specimens collected by the Taiwanese authors.
For molecular analysis, genomic DNA was extracted from whole specimens of Papirioides jacobsoni using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) following the manufacturer's instruction. Before extraction, 1μl of carrier RNA was add into buffer AL. The extracted DNA was eluted in 50 μl elution buffer and stored at -20 °C. Polymerase chain reaction (PCR) for the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the DNA barcode for animals, was conducted using the primers LCO1490 and HCO2198 (Folmer et al. 1994) in a 20-μl volume containing 0.2 mM dNTP, 0.5 μM of each primer, 1.5 mM MgCl 2 , 1.28 μg/μl BSA and 1 U Taq polymerase. Amplification was carried out with a preheat at 94 °C for 1 min, followed by 5 cycles of 94 °C for 30 sec, 45 °C for 30 sec and 72 °C for 50 sec and then by 35 cycles of 94 °C for 30 sec, 51 °C for 30 sec and 72 °C for 50 sec, with a final extension at 72 °C for 10 min. PCR products were checked using 1.5% agarose gel electrophoresis and sequenced by Genomics (Taipei, Taiwan) using an ABI 3730X Genetic Analyzer (Applied Biosystems, CA, USA). DNA sequences were assembled in Geneious (Dotmatics, MA, USA), double-checked by eye and deposited in GenBank under accession numbers ON602032-ON602038.

Discussion
This study is the first update of Collembola in Taiwan in more than 40 years since Chi (1981) listed 26 species in his comprehensive review. The revised checklist comprises 58 species belonging to 31 genera and 12 families, including 13 newly-recorded species, and has been used to update the Catalog of Life in Taiwan database (TaiCoL; taibnet.sinica.edu.tw). Compared to the previous checklist by Chi (1981), this list recognises four more families, including Paronellidae, Dicyrtomidae, Katiannidae and Sminthurididae and follows the most updated taxonomy for genus assignment. This comprehensive checklist serves as an overview of our most up-to-date understanding on the status of collembolan diversity and ecology in Taiwan, fills a knowledge gap resulting from the lack of taxonomic expertise for more than 40 years and provides a foundation for future collembolan studies.
Our results rejected the hypothesis that the "spotty" and "milky" colour-morphs of Papirioides jacobsoni represent two distinct species and concluded that these morphological variations are intraspecific. A possible explanation for the distinct colour-morphs is sexual dimorphism. However, because the voucher specimens used for DNA extraction have become unsuitable for proper morphological examination, we are unable to test this hypothesis. In our phylogenetic results, the species consists of two genetically distinct lineages that are also geographically separated. The mean p-distance between the two lineages is smaller than the interspecific distances between Figure 5. DNA barcode tree of Papiriodes jacobsoni in Taiwan based on neighbor-joining analysis and Kimura's two-parameter model. The specimens analyzed form two genetically distinct lineages, L1 and L2 (A), corresponding to populations in northern and central Taiwan, respectively. Two color-morphs, "spotty" (B) and "milky" (C), can be found in both lineages. Specimens are labels with their NTUM catalog numbers followed by a symbol denoting their color-morphs. Numbers around nodes are bootstrap values. sister species (Porco et al. 2012;Katz et al. 2015). Thus, we consider the genetic variations observed in our samples as intraspecific. Further research with additional samples is needed to understand the morphological polymorphism, genetic structure and phylogeography of this species in Taiwan.
Our field sampling was not conducted systematically. The samples we collected are mostly from the northern part of Taiwan. We also did not attempt to revisit documented locations from which the recorded species were collected in the past. Thus, we were unable to make any specific inference regarding temporal changes based on our study and previous reports. However, we can safely assume that land-use changes in the last several decades have dramatically changed the landscapes and it is likely that habitats in most documented locations have been dramatically altered. It is unclear whether any of the specimens Chi (1981) examined still exist; if they do, the specimens need to be re-examined to confirm their species identity.
The majority of the 13 species newly recorded in this study are large-bodied, atmobiotic (surface-active) species (Potapov et al. 2016), which are relatively easy to find in the field with the naked eye during a targeted search, to collect using an aspirator and to examine and store in the laboratory. Other than the 13 species, many specimens we collected and examined so far could be assigned only to a subfamily or a genus. These putative species are in the families Neanuridae, Onychiuridae, Neelidae, Tomoceridae, Isotomidae, Orchesellidae, Paronellidae, Entomobryidae, Sminthurididae, Arrhopalitidae, Sminthuridae, Bourletiellidae and Dicyrtomidae and their image records are accessible on the lead author's Flickr page (https://flic.kr/ps/3UjMUB). Many of these presumptive species have voucher specimens archived at the NTU Museum of Zoology (preserved in 85% ethanol and stored at 4 °C). These specimens need to be further examined and barcoded to provide a more robust picture of the diversity of Collembola in Taiwan. In fact, the number of species in Taiwan, 58, is relatively low compared to those in neighbouring countries (e.g. 407 in Japan (Hishi et al. 2019)). This low number of species recorded has apparently resulted from the lack of research, as demonstrated by the 40-plus-year gap between Chi's (1981) review and this study.
Using digital photographs for collembolan species identification, albeit unconventional, is an overlooked and under-appreciated avenue that, when used properly, can accelerate the discovery of local species diversity and improve our understanding on the global distribution of widespread species. The combination of digital photography, community science and social media platform (e.g. Collembola of Taiwan Facebook group) has become instrumental in helping us locate certain species in Taiwan and uncover morphological polymorphism in Papirioides jacobsoni. We acknowledge that this approach, in general, has lower accuracy in species-level identification than conventional methods, even for large-bodied species and needs to be used with caution to avoid misidentification. Additionally, its use is likely limited to large-bodied and surface-active species, as smaller species and species living in the soil are less noticeable to the general public, harder to photograph and impossible to identify without examining detailed morphological characters (e.g. chaetotaxy) under a microscope.

Conclusions
Fifty-eight species of Collembola belonging to 31 genera and 12 families have been reported in Taiwan, including 13 species newly recorded in this study. These numbers mark a 123% increase in species richness from the previous comprehensive review. The results of this study have been used to update the "Catalog of Life in Taiwan" (taibnet.sinica.edu.tw) and the species information in the "Checklist of the Collembola of the World" (collembola.org). Additionally, although the dicyrtomid species Papirioides jacobsoni was shown to comprise two divergent mitochondrial lineages, these lineages are not concordant with morphological differences in colour morphs. Finally, we highlighted the potential and limitation of using macro photographs to reach species-level identification in Collembola.