DNA barcodes reveal population-dependent cryptic diversity and various cases of sympatry of Korean leptonetid spiders (Araneae: Leptonetidae)

www.nature.com/scientificreports OPEN DNA barcodes reveal population‑dependent cryptic diversity and various cases of sympatry of Korean leptonetid spiders (Araneae: Leptonetidae) Jong‑Hwa Oh1, Sora Kim3,4* & Seunghwan Lee1,2* Leptonetidae are tiny, rarely encountered spiders that mainly inhabit moist environments, such as caves, leaf litter, and rock piles. Because they are microhabitat specialists, most leptonetid species have short-range endemism, and rarely occur in sympatry. Their small size, relatively simple habitus features and reproductive organ structure increase the difficulty of identification. The identification of leptonetids and other spiders may also be time-consuming due to their sexual dimorphism, polymorphism, and lack of diagnostic characteristics in juveniles. DNA barcoding has been used as an effective tool for species identification to overcome these obstacles. Herein, we conducted a test of DNA barcoding based on 424 specimens of Korean Leptonetidae representing 76 morphospecies. A threshold of 4.2% based on maximum intraspecific genetic divergence was estimated to efficiently differentiate the morphospecies. The species assignments tested by five species delimitation methods (ABGD, ASAP, GMYC, PTP, and bPTP) were consistent with the morphological identifications for only 47 morphospecies (61.8%), indicating many cases of cryptic diversity among the remaining morphospecies. Furthermore, sympatry in leptonetids, which are known to be rare, was revealed to be common in South Korea, especially in epigean species. Our results showed that sympatries within families, congeners, and intraclades potentially occur throughout the entire region of Korea. Spiders of the family Leptonetidae Simon, 1890 currently include 21 genera and 368 species that mainly inhabit the Holarctic region. To date, 57 species in seven genera have been recognized in the Nearctic region, 74 species in eight genera have been identified in the Mediterranean region, and 237 species in eight genera have been identified in A sia1. Leptonetids are small (1–3 mm) spiders that build sheet webs, on which they hang below. They are known to prefer dark and moist microhabitats, such as leaf litter, layered rock piles, mines, and caves2–7. This family is well-known to have a strong cave association because more than 50% of the species have only been identified in cave habitats. Many of these cave species have highly troglomorphic morphologies, such as eye reduction, depigmentation, and elongation of the a ppendages8. In contrast, in South Korea, only 17% (9 species out of 52 species) of the species have only been identified in caves, and the remainder have been identified in epigean habitats2,3,7,9–23. Subterranean habitats and islands are known as one of the best barriers to geographic isolation, resulting in high endemism in arthropod taxa, including s piders24–30. However, within leptonetids, especially within epigean species, the speciation barrier is ambiguous and poorly understood. Because they are rarely seen, the general biology and life history of leptonetid spiders are poorly understood, and only some of the reproductive biology has been reported thus f ar4,30,31. Due to their habitat preferences, most species have a limited distribution range and rarely occur in sympatry, with this characteristic only observed in a few epigean p opulations4,5. In studies of North American leptonetids, Gertsch suggested intraspecific polymorphism in troglomorphic characters, such as the reduction of eyes and depigmentation in Neoleptoneta capilla32. Additionally, Tayshaneta 1 Laboratory of Insect Biosystematics, Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea. 2Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea. 3Laboratory of Insect Phylogenetics and Evolution, Department of Plant Protection & Quarantine, Jeonbuk National University, Jeonju, Republic of Korea. 4Department of Agricultural Convergence Technology, Jeonbuk National University, Jeonju, Republic of Korea. *email: ; Scientific Reports | (2022) 12:15528 | https://doi.org/10.1038/s41598-022-18666-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Map with sampling sites for collected specimens used in the present study. Pie charts indicate the proportion of each genus sampled from respective administrative localities. The abbreviations of each administrative district follow: GG, Gyeonggi-do; GW, Gangwon-do; CB, Chungcheongbuk-do; CN, Chungcheongnam-do; JB, Jeollabuk-do; JN, Jeollanam-do; GB: Gyeongsangbuk-do; GN, Gyeongsangnam-do; JJ, Jeju-do. The map was prepared using QGIS 3.22.1 (https://www.qgis.org/ko/site/). myopica and Tayshaneta paraconcinna presented more complex polymorphisms and genetic diversity that was dependent on the population and habitat t ype6,33. Similar patterns were also found in South Korea, but only between epigean populations. Compared with research performed in North America, studies performed on Korean leptonetids have focused on α-level diversity and treated cases of polymorphism as a separate species2,3,7,22,23. Sexual dimorphism, polymorphisms, and limited morphological information on juveniles or larvae have been obstacles for morphological identification34,35. To overcome these obstacles, DNA barcoding has been developed, and it represents an efficient and popular tool for taxonomic investigations for the accurate species identification, cryptic species discovery and biodiversity estimates of animal t axa36–44, including s piders45–62. DNA barcoding of leptonetids has also been performed based on genetic distance and the Automatic Barcode Gap Discovery (ABGD) method in Leptonetela taxa62. Their study included numerous species from over a hundred cave populations throughout China and Europe. However, their sampling was mainly based on populations from caves and did not include populations from epigean habitats. In this study, we (i) tested the utility of DNA barcodes for species identification, delimitation, and new species discovery, (ii) compared morphology-based species delimitation and barcode-based approaches, (iii) searched for types of barriers that lead to genetic diversity and speciation, including epigean leptonetids, by determining the range of species distribution, (iv) provided morphological characters that support results of delimitation methods, (v) detected cases of cryptic species, and (vi) generated a DNA barcode reference library of described species and putative new leptonetid species. Material and methods Taxon sampling and morphological identification. As part of an ongoing revision of the Korean Lep- tonetidae, we examined approximately 900 specimens (including more than 600 adult specimens) collected from approximately 150 sites in 15 (out of 17) administrative districts, including 22 caves/mines and 11 islands of South Korea (Fig. 1, Supplementary Table S1 (...truncated)