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DNA barcodes revealed population dependentcryptic diversity and various sympatry of Koreanleptonetid spider (Araneae: Leptonetidae)Jong-Hwa Oh 

Seoul National UniversitySora Kim  ( skim01@jbnu.ac.kr )

Jeonbuk National UniversitySeunghwan Lee 

Seoul National University

Article

Keywords:

Posted Date: March 28th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-1480354/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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AbstractLeptonetidae are tiny and rarely encountered spiders, mainly inhabiting moist environments such ascaves, leaf litter, and rock piles. Because they are microhabitat specialists, most species are known tohave short range endemism, rarely occurring in sympatry. Due to their small size, relatively simplefeatures of habitus and structure of reproductive organs made it challenging on su�cient identi�cation.Not only leptonetids, but also identi�cation in other spiders having sexual dimorphism, polymorphism,and lack of diagnosable keys in juvenile made it even more time consuming. To overcome theseobstacles, DNA barcoding has been used as an effective tool for species identi�cation. Herein, weconducted a test of DNA barcoding with Korean Leptonetidae based on 422 specimens representing 76morphospecies. A threshold of 4.2% genetic divergence was estimated to differentiate the morphologicalspecies e�ciently. The species assignments tested by �ve species delimitation methods (ABGD, ASAP,GMYC, PTP, and bPTP) were consistent with morphological identi�cations for 49 species (64.5%),indicating many cases of cryptic diversity. Further, sympatry in leptonetids was revealed to be common inSouth Korea, especially in epigean species. Our results showed sympatries no matter within family,congeners, and interclade potentially occurring throughout the entire region of the Korea.

IntroductionSpiders of the family Leptonetidae Simon, 1890 are relatively small (1-3mm) members. The familyincludes 20 genera and 366 species around the world, so far, of which 57 species in six genera in Northand Central America, 73 species in eight genera in Mediterranean, and 236 species in seven genera arerecognized in Asia1.

Leptonetids build sheet-webs on which they hang below. They are known to prefer dark and moistmicrohabitats such as leaf litter, layered rock piles, mines, and caves2–7. They are one of the best-knownspiders for cave association and more than 50% of the species are known only from caves. Many ofthese cave species have highly troglomorphic morphologies such as eyes reduction, depigmentation, andelongation of the appendages8. Oppositely in South Korea, only 17% (9 species out of 52 species) of thespecies are known only from caves, and the rest of them are known from epigean habitat2,3,7,9−23.Subterranean habitats and islands are known as one of the best barriers for geographic isolationresulting in high endemism in arthropod taxa including spiders24–30. However, upon leptonetids,especially in epigean species, speciation barrier is ambiguous and poorly known. Because they are rarelyseen, general biology and life history of leptonetid spiders is poorly known, and only some of reproductivebiology has been reported, so far4,30,31. However, due to their habitat preferences, most species have alimited distribution range, rarely occurring in sympatry, only found in a few epigean populations4,5.

Studies of North American leptonetids, Gertsch suggested intraspeci�c polymorphism with troglomorphiccharacters such as reduction of eye and depigmentation in Neoleptoneta capilla 32. Also, Tayshanetamyopica and Tayshaneta paraconcinna have been discovered in more complex polymorphisms with

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genetic diversities depending on populations and type of habitats6,33. Similar patterns were also found inSouth Korea, but only between epigean populations. Unlike studies from North America, studies of Koreanleptonetids have focused on α-level diversity, treating cases of polymorphism as a separatedspecies2,3,7,22,23.

Sexual dimorphism, polymorphism, and lack of morphological information on juveniles or larva has beenan obstacle for morphological identi�cations34,35. To overcome these obstacles, DNA barcoding has beendeveloped as an e�cient tool and has become one of the most popular tools for taxonomic approachesfor accurate species identi�cation, cryptic species discovery, and biodiversity studies in animal taxa36–44,including spiders45–62. DNA barcoding of leptonetids have also been performed based on geneticdistance and Automatic Barcode Gap Discovery (ABGD) method in Leptonetela taxa62. Their studyincluded numerous species from over hundred cave populations throughout China and Europe. However,their sampling was mainly based on populations from caves, without including populations from epigeanhabitats.

In this study, our aim was to: (i) test the utility of DNA barcode for species identi�cation, delimitation, andnew species discovery, (ii) compare between morphological identi�cation and species delimitationmethods, (iii) search for types of barriers that led to speciation including epigean leptonetids bydetermining the range of species distribution, (iv) provide morphological characters that supports resultsof delimitation methods, (v) detect cases of cryptic species, and (vi) provide a DNA reference library ofdescribed species, and putatively new leptonetid species.

Material And Methods

Taxon sampling and morphological identi�cationAltogether, 411 individuals were collected from 102 sites of 14 administrative districts, including 22caves/mines and 11 islands of South Korea (Fig. 1, Supplementary Table S1 online). Samples weremainly collected by net sifting, pit-fall traps, and exploration in caves or mines. Between 1–26 individualswere sampled from each site, placed directly into 98% ethanol, storage in -20°C. Before DNA extraction,habitus and the male palp of the specimens were examined under Leica Z16 APO stereomicroscope.Female genitalia were separated from opisthosoma using microsurgery stiletto knife. Separated genitaliawere cleared by heating in 5ml tubes of lactic acid or 10% KOH solution for 1hour to dissolve extraneoustissue, and then examined and photographed with Olympus BX53 compound microscope. All specimenswith vouchers are deposited in the College of Agriculture and Life Sciences, Seoul National University(CALS, SNU, Seoul), National Institute of Biological Resources (NIBR, Incheon), and Yangpyeong InsectMuseum (YIM, Yangpyeong), Republic of Korea.

DNA extraction, PCR ampli�cation and Sequencing

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Genomic DNA was extracted from a tissue by grinding usually 2–4 legs, or whole body except abdomenusing DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany) following the manufacturer’s protocols.For PCR ampli�cations for mitochondrial cytochrome oxidase subunit I (mtCOI, ~ 901bp), we useduniversal primers or primers developed and used in arachnid taxa. Primer combinations we used in thisstudy were forward LCO1490 (5’-GGTCAACAAATCATCATAAAGATATTGG-3’)63 with reverse HCO2568 (5’-GCTACAACATAATAAGTATCATG-3’)64 or HCOoutout (5’-GTAAATATATGRTGDGCTC-3’)65. We also tried toamplify internal transcribed spacers (ITS1, ITS2) using primer pair including forward CAS18sF1 (5’-TACACACCGCCCGTCGCTACTA-3’) with reverse CAS28sB1d (5’-TTCTTTTCCTCCSCTTAYTRATATGCTTAA-3’) for ITS units 1 and 266, forward ITS5.8 (5’-GGGACGATGAAGAACGCAGC-3’) with reverse ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) for ITS unit 267. Ampli�cation was performed using AccuPower PCRPremix (Bioneer, Daejeon, Republic of Korea) following the standard protocols. For COI region, the PCRcondition consisted to initial denaturation at 95°C for 2min, followed by 35 cycles of denaturation 95°Cfor 30s, annealing at 45–50°C for 30s, extension at 72°C for 45s, and a �nal extension at 72°C for 10min.The annealing temperature was adjusted to 40–55°C to ITS region, with 30–40 cycles. However, weachieved remarkably low success rate both in ITS units 1 and 2 ampli�cation. Therefore, they werediscontinued from our further analysis. Successfully ampli�ed PCR products were checked in 1.2%agarose gels, and were puri�ed and sequenced at BIONICS, Inc. (Seongdong-gu, Seoul, Republic ofKorea).

Sequence analysis, genetic distance, Phylogenetic analysis411 COI raw sequences were assembled and edited using SeqManTMII (version 5.01, 2001; DNA-star™).We eliminated poor quality of DNA and short sequences in order to prevent any risk of confusion orerrors. 11 additional sequences were downloaded from NCBI. Therefore, a total of 422 sequences werecarried out for alignment using MAFFT version 768 through the EMBL-EBI online portal using the L-INS-imethod. The sequences were deposited in GenBank (ON041801–ON042211, Supplementary Table S1).The sequence data were then combined using SequenceMatrix windows ver. 1.869. To calculateintra/interspeci�c pairwise genetic distances, we implemented Kimura-2 parameter (K2P) model usingMEGA 7.070. Finally, haplotype data were generated in DnaSP6.1271 to identify the distinct haplotypes.

For phylogenetic analysis, Neighbor-Joining (NJ), Maximum likelihood (ML), and Bayesian Inference (BI)were generated in order to test monophyletic criteria of species delimitation. Aligned sequences werecarried out to PartitionFinder v2.1.172 to choose the best �t model for the analysis. The NJ tree wasconstructed using MEGA 7.0 under K2P model70. RaxML GUI v2.0 was used under GTR + GAMMA modelfor ML tree analysis73,74. BI tree was constructed using MrBayes v3.2.775. The Markov chain Monte Carlosearch for the data matrix ran four chains for 10,000,000 generations with 25% burn-in, and samplingevery 100 generations. Finally, the phylogenetic tree was visualized in FigTree v1.4.476.

Barcoding, species delimitation, and MOTUs estimation.

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To estimate the number of Molecular Operational Taxonomic Units (MOTUs), we used �ve speciesdelimitation methods: Automatic Barcode Gap Discovery (ABGD)77, Assemble Species by AutomaticPartitioning (ASAP)78, Generalized Mixed Yule-coalescent (GMYC)79, Poisson-Tree-Processes (PTP), andBayesian implementation of the PTP (bPTP)80.

ABGD method delimit hypothetical species by calculating pairwise distance based on barcode gap. TheABGD analysis was performed online (https://bioinfo.mnhn.fr/abi/public/abgd/) under Jukes-Cantor(JC69), Kimura 2-parameter (K2P), and uncorrected distance (p-distance) model with relative gap width(X = 1.5).

ASAP is a recently developed method designed to propose species partitions using hierarchical clusteringalgorithm based on pairwise genetic distances. The aligned sequences were submitted online(https://bioinfo.mnhn.fr/abi/public/asap/) under models same with ABGD method in default settings.

In GMYC analysis, we used BEAST v2.6.681 to obtain ultrametric tree, under a strict molecular clockmodel. On the prior, we used Yule speciation model running 20 million generations, sampling every 1000generations. We further checked for stationarity and determined burn-in using TRACER v1.7.282, and thendiscarded as 15% burn-in, 0.5 posterior probability using TreeAnnotator81. For GMYC analysis, ultrametrictree were then carried out in RStudio (https://www.r-project.org/) using the “splits” package83.

PTP is a coalescent-based delimitation method that requires phylogenetic input tree, and bPTP is anupdated version of the original PTP, adding Bayesian support (BS) values to delimited species on theinput tree. For both PTP and bPTP analysis, we employed BI tree as input tree, implemented online(https://species.h-its.org/ptp), running 100000 MCMC generations, with a thinning of 100, burn-in of 0.1,and removing the outgroups for improved results.

ResultsMorphological identi�cation.

Based on the morphological examination, 75 morphospecies including 12 new species and 23 putativenew species in four genera (Falcileptoneta, Leptoneta, Longileptoneta, and Masirana) of leptonetids wereidenti�ed from 409 specimens used in this study (Supplementary Table S2 online). Additionally, twoindividuals of newly recorded family, Telemidae from South Korea was also included as an outgroup.

Although leptonetids are known as microhabitat specialists and highly localized, many species,especially in epigean leptonetids, were sampled from various distant populations, indicating that they aremore widely distributed then expected around the Korean peninsula (Falcileptoneta odaesanensis,Leptoneta taeguensis, Leptoneta chilbosanensis, Leptoneta spinipalpus, Longileptoneta weolakensis,Longileptoneta songniensis, Longileptoneta sp3). In some cases, the same species were distributedacross the islands (Falcileptoneta sp17, Falcileptoneta sp21, Leptoneta namhensis, Leptonetapaikmyeonggulensis).

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Genetic distance divergence of species identi�cation.

Haplotype data analysis revealed 200 distinct haplotypes (Supplementary Fig. S1 online). The averagevalue of K2P genetic divergence was 20.74% across all specimens of the dataset. Intraspeci�c geneticdivergence of K2P were 1.27% (ranging from 0–18.41%), with approximately 13 times increased withincongeners which is 16.64% (ranging from 14.49 to 21.22%) on average (Table 1). However, the divergenceamong each congener pair varied from 10.46%, to 21.22%: Falcileptoneta (Mean = 20.40%),Longileptoneta (Mean = 10.46%), Leptoneta (Mean = 21.22%), and Masirana (Mean = 14.49%). Detailedvalues of each genera are provided in Table 2.

In the results of the analysis to investigate a threshold in order to evaluate number of MOTUs inLeptonetidae, the maximum value of intraspeci�c K2P genetic divergence was less than 4.2%, and 11morphospecies were detected having over 4.2% of maximum intraspeci�c divergence (Fig. 3). 

 Table 1

K2P genetic distance depending on species and genuslevel of Leptonetidae

Comparisons Mean of K2P Genetic Distance (%)

Overall 20.74

Within species 1.27

Within congeners 16.64

 Table 2

K2P genetic distances between species within each genus.Comparisons Mean (%) Minimum (%) Maximum (%)

Falcileptoneta 20.40 4.50 31.0

Longileptoneta 10.46 3.60 17.10

Leptoneta 21.22 9.70 29.20

Masirana 14.49 11.10 16.80

MOTUs estimation.

The species delimitation methods of ABGD, ASAP, GMYC, PTP and bPTP, yielded 92, 98, 112, 117, and120 of MOUTs, respectively. Color bars in the ML tree indicate the results of delineated MOTUs bydifferent methods (Fig. 2). Unlike morphological identi�cation, species delimitation methods mostlydelineated depending on population level rather than morphological appearances (see Supplementary

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Table S3 online). Phylogenetic tree-based methods especially tend to over-split than genetic distance-based methods. As a result, 49 MOTUs which are 64.5% of the total agree with morphologicaldelimitation of species.

ABGD analysis on each JC69, K2P, and p-distance substitution model produced non-identical MOTUs,resulting in 82, 92, and 91 MOTUs respectively with all P = 0.035938 (for more details, see SupplementaryTable S4 online). Six morphospecies, Falcileptoneta secula, Falcileptoneta maewhaensis, Falcileptonetasp2, Falcileptoneta odaesanensis, Falcileptoneta sp17, and Leptoneta chilbosanensis were divided intotwo, Falcileptoneta chiakensis into �ve, and Leptoneta taeguensis into six MOTUs by ABGD. On the otherhand, Longileptoneta weolakensis and Leptoneta spinipalpus were observed to share a single MOTU.

Unlike ABGD method, ASAP analysis produced identical MOTU results on each JC69, K2P, and p-distancesubstitution model, all yielding 98 MOTUs with 7.00, 9.50, and 6.50 of ASAP-score, respectively (for moredetails, see Supplementary Table S5 online). Compared with ABGD, additional MOTUs were observed inseven species: Falcileptoneta chiakensis, Falcileptoneta hansanensis, Longileptoneta gayaensis,Longileptoneta weolakensis, Leptoneta spinipalpus, Leptoneta namhensis, and Masirana ilweolensis.

GMYC analysis produced more MOTUs, likely over-splitting into 112 species. Unlike ABGD and ASAPmethods, 12 species were divided additional MOTUs by the GMYC method: Falcileptoneta sp2,Falcileptoneta sp3, Falcileptoneta sp4, Falcileptoneta juwangensis, Falcileptoneta geumsanensis,Falcileptoneta unmunensis, Longileptoneta byeonsanbando, Longileptoneta sp3, Longileptoneta sp4,Longileptoneta sp6, Leptoneta taeguensis, and Leptoneta paikmyeonggulensis. Conversely,Falcileptoneta hansanensis was recovered as a single MOTU in GMYC analysis.

PTP analysis yielded 117 species, delineating similarly with GMYC. However, more MOTUs were observedin �ve species: Falcileptoneta geumsanensis, Falcileptoneta hansanensis, Longileptoneta sp4, Leptonetasp1 and Leptoneta paikmyeonggulensis. Additionally, Falcileptoneta juwangensis was recovered as asingle MOTU from GMYC.

bPTP produced the most MOTUs among the methods we used in this study, yielding 120 species.Although bPTP recovered Leptoneta sp1 into single MOTU from PTP method, it divided Falcileptonetaunmunensis into three, Leptoneta paikmyeonggulensis into four, and Masirana ilweolensis into �veMOTUs.

Detection of cryptic species.

The threshold based on maximum intraspeci�c K2P genetic divergence was estimated by 4.2% from thedataset, with 11 morphospecies having over maximum divergence of estimated threshold (5.8–18.4%)(Fig. 3). In particular, nine morphospecies were delineated into multiple MOTUs by all �ve speciesdelimitation methods.

Compared to morphological results, species delimitation methods mainly resulted in delineating theMOTUs depending on population level, especially on the epigean leptonetids. Firstly, based on epigean

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leptonetids (Fig. 4), the species Leptoneta taeguensis were divided into six to seven (Fig. 4a),Falcileptoneta odaesanensis into two (Fig. 4b), and Falcileptoneta chiakensis into �ve to six (Fig. 4c)MOTUs in species delimitation methods used in this study, depending on each locality (for morphologicaldetails, see supplementary Fig. S2 online). Secondly, in troglophilic leptonetids (Fig. 5), separated MOTUswas detected between cave populations and epigean populations even though those populations werenot readily diagnosable (Falcileptoneta secula and Falcileptoneta maewhaensis) (Fig. 5a, b, seesupplementary Fig. S3 online for morphological details). Finally, species that are geographically isolatedby islands showed both single and double MOTUs by all �ve species delimitation methods (Fig. 5c, seesupplementary Fig. S3 online for morphological details).

Wide distributed species, and congeneric sympatry in leptonetids.

Firstly, many cases mainly members of the genus Longileptoneta showed that a single MOTU weresampled between distant populations indicating they are distributed in a large range, potentially havingsympatry within congeners (Fig. 5a): (i) The species in Longileptoneta which are potentially a newspecies are distributed nationwide in the western part of South Korea (Pocheon-si, Jaecheon-si, Daejeon,Haenam-gun). (ii) Longileptoneta songniensis which are known only from Mt. Songnisan (Boeun-gun)was also found on Ganghwado island (Incheon). (iii) Longileptoneta weolakensis which is previouslyknown only from Mt. Weolaksan (Jecheon-si) was also found on Mt. Baegunsan (Pocheon-si).

Secondly, some cases revealed that different populations which are across the islands were sharing asingle MOTU. (i) Falcileptoneta sp17 from Mt. Duryunsan (Haenam-gun) and Wando island (Wando-gun),but different from Geogumdo island (Goheung-gun) (Fig. 5c), (ii) Falcileptoneta sp21 from Namsan Park(Goseong-gun) and Namhae island (Namhae-gun), (iii) Longileptoneta songniensis from Mt. Songnisan(Boeun-gun) and Ganghwado island (Incheon), (iv) Leptoneta paikmyeonggulensis from Namhae islandand Dolsan island (Yeosu-si) resulted in a single MOTUs, respectively.

Finally, two or more species of epigean populations belonging to the genus Falcileptoneta found at thesame locality were detected (Fig. 6b): (i) F. chiakensis and Falcileptoneta sp2 from Mt. Chiaksan (Wonju-si), (ii) Falcileptoneta umyeonsanensis and Leptoneta chilbosanensis in Mt. Kwanaksan (Seoul), (iii)Falcileptoneta odaesanensis, Falcileptoneta sp4 and Leptoneta chilbosanensis in Mt. Seoraksan (Inje-gun), (iv) Falcileptoneta hansanensis and Falcileptoneta sp23 from Mt. Mireuksan (Tongyeong-si), (v)Falcileptoneta geumsanensis, Falcileptoneta sp21 and Falcileptoneta sp22 in Namhae island (Namhae-gun), (vi) Falcileptoneta sp8 and Falcileptoneta sp17 from Wando island (Wando-gun). However, in cavehabitats, none of the sympatry was detected.

Sympatry in interclade species.

The clade that include Falcileptoneta sp2, Falcileptoneta sp3, Falcileptoneta umyeonsanensis,Falcileptoneta sp4, Falcileptoneta sp5, Leptoneta kwangreungensis, and Leptoneta chilbosanensis has asharp tooth-like tibial apophyses in common. Identi�cation between those species is quite challenging,but they can be diagnosed by the thickness and length of the tibial apophysis of the male palp, spotted

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patterns of abdomen, and length of the body size. Most of the species appears to be in short-rangeendemism and been only found in a small range of habitat (one or two mountain ranges). However, thepopulation from Mt. Surisan (Ansan-si) were sharing a single MOTU with one individual fromFalcileptoneta umyeonsanensis population (Mt. Kwanaksan, Seoul), one individual from Falcileptonetasp4 population (Mt. Seoraksan, Inje-gun), indicating that they are potentially distributed in at least thewhole of the central region of the Korean peninsula, sharing habitat with numerous populations (Fig. 7).

Biogeographical patterns.

Our phylogenetic results suggest that leptonetids are mainly distributed in small ranges, resulting in anarrow endemism. However, many species, especially in the genus Longileptoneta, were shown to bedistributed in larger range, with overlapped range with other species. Also, presence of sympatry wasdetected all within family level, congeners, and interclade species. Additionally, phylogenetic analysesrevealed that species which are genetically close together were mainly geographically close.

DiscussionsAccurate identi�cations of leptonetid species can be challenging due to their small size, simple habitusand structure of the male palp. Likewise, morphological identi�cation with female and juvenile specimensis also problematic due to lack of morphological information and undiagnosable genitalia4,5,33.Therefore, we tested the utility of DNA barcodes in species identi�cation based on maximum intraspeci�cdivergences and �ve types of species delimitation methods with Korean Leptonetidae. Our resultssuggest that a maximum value of 4.2% could serve as an e�cient standard for preliminary speciesdelimitation within Korean Leptonetidae. Additionally, species delimitation results showed that methodsof ABGD, ASAP, GMYC, PTP, and bPTP yielded 92, 98, 112, 117, and 120 MOTUs, respectively (Fig. 2).Phylogenetic tree-based delimitation methods tend to be more sensitive to split more MOTUs comparedto barcode gap-based methods (Supplementary table S3 online). Based on methods we used in thisstudy, cryptic diversity was detected in 11 morphospecies, especially with seven morphospeciesdelineated by population level (Fig. 4, 5). Those cryptic species cannot be readily distinguished bymorphology, yet genetic data may suggest that they are on different evolutionary trajectories. Crypticspecies are important to add our understanding of biodiversity, connecting between taxonomy andphylogenetic studies, and evolution 84.

Leptonetids are known to be dispersal-limited spiders, preferring speci�c type of microhabitat such asleaf litter, caves, and mines, creating distributional patterns as ‘narrow endemism’5,33. As demonstrated inmany other arachnid lineages, the biological traits of restricted dispersion ability and high microhabitatspecialization generally lead to biogeographic histories dominated by vicariance, with few dispersalevents85–87. Islands and caves play a role in vicariance, causing high endemism in arthropod lineages88–

92. Throughout this study, we newly found that not only a considerable number of leptonetid species haveexpanded distributions, being found beyond restricted zones such as caves and islands, but also sharinghabitat no matter between clade, species and genus level (Fig. 6, 7). Among them, four cases revealed

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that populations occurring both in mainland of Korea and the islands were sharing a single MOTU,raising the question: How did they get there? We conclude that there are two potential reasons for thispattern. First, leptonetids may have a ballooning behavior, but no ability to balloon a long distance. Unlikeother spiders, there are no records of ballooning behavior in leptonetids, so far. Based on the habitatpreference, we can infer that they are sensitive to humidity, and fatal when ballooning due to lowhumidity in the air than litter or caves, resulting in short distance of ballooning. Second, speciation hasnot occurred after geographic isolation, yet. Localities where single MOTUs of the leptonetids occur bothin mainland and the islands have a low sea level between. Therefore, if leptonetids cannot balloon, wecan predict that the islands were isolated very recently and that speciation has not yet taken place.

Single morphospecies between cave population and epigean population nearby the cave resulted inmultiple MOTUs in the species delimitation methods we used in this study (Fig. 5a, b). Ledford and hiscolleague discussed similar cases in Tashaneta species based on multigene phylogeny, treating thesecases as intraspeci�c polymorphisms even though of furcation on phylogenetic tree33. However, in onecase in this study, F. simboggulensis which are known as troglobitic spiders in Simboggul cave, wassharing a single MOTU with epigean individual nearby the cave indicating gene �ow occurs between thiscave and epigean populations around the cave. Simboggul cave is one of the exceptional caves beingaffected by external environments in temperature (Pers. Comm. Y.G. Choi). This suggests that gene �owsbetween cave and epigean populations could be more related with temperature than darkness of thecave.

Some morphospecies were highly genetically distant between populations (Falcileptoneta secula,Falcileptoneta odaesanensis, Falcileptoneta maewhaensis, Falcileptoneta sp17, and Leptonetataeguensis) (Fig. 4–5, Supplementary Fig. S2, 3). We �gured out that most of those species belong to thegenus Falcileptoneta. Compared to other Korean leptonetid genera, especially Longileptoneta, species inFalcileptoneta tend to have more dispersal limited endemism. Our sampling data around South Koreaindicated that, in most cases, many individuals of Falcileptoneta species were sampled in small ranges,while few individuals of Longileptoneta species were sampled but in larger ranges than expected(Fig. 6a). Based on the analysis of the sampling data we identi�ed the phenomenon that species in thegenus Longileptoneta mainly tend to spread out, while Falcileptoneta gather. Especially, species sampledfrom natural caves that cause narrow endemism, were all in the member of the genus Falcileptoneta.

In traditional taxonomy of spiders, structures of the female genitalia and the male palp have been key forspecies identi�cation. However, many cases in our result showed that species that are undistinguishablebased on the male palp belong to different MOTUs. Rather, the shape of sternum, patterns in abdomen,and the ratio between length of the body and leg were somewhat diagnosable from dependent MOTUs.Although some of the cases had sexual dimorphism of the shape of sternum, at least in same sex had asame shape (Supplementary Fig. 2, 3).

In our study, we include several species in the genus Leptoneta. However, phylogenomic andbiogeographic study of the family Leptonetidae shows that Leptoneta are restricted in Mediterranean

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Europe, and all of the species in Leptoneta are morphologically, and geographically misplaced5 (Ledfordet al, 2021). Although Seo transferred many species of Leptoneta to Falcileptoneta (2015)18, more thanten species of Korean leptonetids are still remaining in this genus. Based on our morphological andphylogenetic analysis of maintained Korean Leptoneta, we agree that species in this genus are allmisplaced, and Korean leptonetid should be revised.

DeclarationsData Availability

Accession Codes: The COI sequences generated and analyzed during the current study are available inthe Genbank repository, from ON041801 to ON042211.

Acknowledgements

We thank Minho Lee, Duk-Young Park, Minseok Oh & Jaeseok Oh (Department of AgriculturalBiotechnology, Seoul National University, Seoul, Korea), Sungwon Yoon (Department of Forest LandscapeArchitecture, Sanji University, Wonju, Korea), Jeong-Hoon Ahn (Goseong Middle School), Jun-Gi Lee &Jun-Ho Lee (College of Agriculture & Life Sciences, Kangwon National University, Chuncheon, Korea),Hyeonjun Shin (Department of Biology, Soonchunhyang University, Asan, Korea) and Jangwon Seo(Department of Biological Science and Biotechnology, Chungbuk National University, Chungju, Korea) fortheir help collecting specimens. We also thank Seung-Ha Hwang (Department of Game, ChungkangUniversity, Icheon, Korea) for assistance in preparing the �gures. The �rst author is especially grateful toYong-Gun Choi (The Korean Institute of Biospeleology, Daejeon, Korea), Dr. Jung Sun Yoo (NationalInstitute of Biological Resources, Incheon, Korea), Jae-Won Kim (Yangpyeong Insectarium, Yangpyeong-gun, Korea) Dongyoung Kim (Department of Biological Sciences, Ajou University) for providingspecimens with sharing valuable comments during this study. Finally, we are grateful to Dr. DimitarDimitrov (Department of Natural History, University of Bergen, Bergen, Norway) for valuable and helpfulcomments on the draft version of the manuscript. This work was supported by a grant from the NationalInstitute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic ofKorea (NIBR202002204, NIBR202102203), and Korea Institute of Planning and Evaluation for Technologyin Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs ConvergenceTechnologies Program for Educating Creative Global Leader Program (or Project), funded by Ministry ofAgriculture, Food and Rural Affairs (MAFRA)(no.321001-03), and the Basic Science Research Programthrough the National Research Foundation of Korea (NRF), funded by the Ministry of Education(NRF2020R1I1A2069484).

Author Contributions

All authors conceived and designed the study; S.K & S.L supported funding acquisition; J.H.O conductedthe sampling, morphological identi�cation, DNA extraction and PCR experiments, performed sequencedata analyses, MOTU analyses, prepared �gures and tables; J.H.O & S.K wrote and revised the

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manuscript; S.K. edited the manuscript; All authors con�rmed and approved the �nal version of themanuscript.

Competing interests

The authors declare no competing interests.

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Figures

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Figure 1

Map with 102 sampling sites for 411 specimens used in the present study. Pie charts indicate theproportion of each genera sampled from respective administrative localities. The abbreviations of eachadministrative districts 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. All maps used in this study was prepared using QGIS 3.22.1(https://www.qgis.org/ko/site/)

Figure 2

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Maximum Likelihood (ML) tree of 422 COI sequences, including morphological analysis and results of�ve different species delimitation methods. Color bars indicate the results of each delimitation methods.In the ML tree, blue panels indicate that all species delimitation methods agreeing with morphologicalidenti�cation, while red panels show one or more methods that disagree.

Figure 3

Maximum intraspeci�c divergences (%) based on Kimura 2-parameter model from the dataset.Morphospecies having single specimen are not included.

Figure 4

Cryptic species detected between various populations of epigean leptonetids. (a) Leptoneta taeguensisfrom (1) Hapcheon-gun, (2) Miryang-si, (3) Yangsan-si, (4) Daegu, (5) Cheongdo-gun, and (6) Ulju-gun; (b)Falcileptoneta odaesanensis from (7) Goseong-gun, (8) Inje-gun, Yangyang-gun, Gangneung-si,Pyeongchang gun, and Hongcheon-gun, (c) Falcileptoneta chiakensis from (9) Danyang-gun, (10) Wonju-si, (11) Pyeongchang-gun, (12) Yeongju-si, and (13) Jaecheon-si. Asterisks indicates type localities ofeach species.

Figure 5

Cryptic species detected between cave populations / epigean populations, and between mainlandpopulations / island populations. (a) Falcileptoneta secula from (14) Jumunjin (Gangneung-si) and (15)Magwihalmigul cave (Gangneung-si), (b) Falcileptoneta maewhaensis from (16) Gaya-myeon (Hapcheon-gun) and (17) Betishale cave (Hapcheon-gun), (c) Falcileptoneta sp17 from (18) Geogumdo island(Goheung-gun) and (19) Mt. Duryunsan (Haenam-gun), Wando island (Wando-gun). Asterisks indicatestype localities of each species.

Figure 6

Maps showing sympatric localities between congeneric species. (a) Longileptoneta, (b) Falcileptoneta.Members of Longileptoneta are mainly wider distributed compared to species of Falcileptoneta,suggesting a possibility that this genus may have potentially more sympatric distributions than expected.

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Figure 7

Interclade sympatry occurring in central region of Korean peninsula. 

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