103 urn:lsid:arphahub.com:pub:77d0745d-c3a1-5248-81de-8cdc02bed84a urn:lsid:zoobank.org:pub:F56F6CF9-7502-4001-A751-35D5F2EF6CA0 Arthropod Systematics & Phylogeny ASP 1863-7221 1864-8312 Senckenberg Gesellschaft für Naturforschung 10.3897/asp.82.e113041 113041 Research Article Crustacea Isopoda Malacostraca Taxonomy Integrative taxonomy reveals exceptional species diversity of Lucasioides from China (Isopoda: Oniscidea: Agnaridae) Wang Jin https://orcid.org/0000-0002-1189-6442 1 Writing - original draft Yao Chong-Hui https://orcid.org/0009-0009-6781-6964 1 Software Jiang Chao https://orcid.org/0000-0003-1841-1169 2 Funding acquisition Li Wei-Chun weichunlee@126.com https://orcid.org/0000-0003-0154-861X 1 Supervision College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, China Jiangxi Agricultural University Nanchang China State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China China Academy of Chinese Medical Sciences beijing China

Corresponding authors: Chao Jiang (jiangchao0411@126.com), Weichun Li (weichunlee@126.com)

 2024 02 07 2024 82 527 549 E3A8F026-F964-5504-980C-341627EF4BE6 B2966C0A-75B7-4B73-A45C-0A2EFB159715 20 09 2023 26 03 2024 Jin Wang, Chong-Hui Yao, Chao Jiang, Wei-Chun Li This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://zoobank.org/B2966C0A-75B7-4B73-A45C-0A2EFB159715

The genus Lucasioides Kwon, 1993 (Isopoda: Oniscidea: Agnaridae) occurs in Asia, but confined only to China, Japan, Korea, and Russian Siberia. The ambiguously morphological differences among some members of the genus make a dilemma: the species recognition, whether morphologically similar or different, is uncertain. In this paper, we present first morphometric and molecular data for the genus from a broad sample. DNA sequences (mitochondrial COI, nuclear 18S rRNA, 28S rRNA, and NaK) were generated and integrated with morphological evidence to reveal the cryptic species and delimit the new species within the genus. Seven species are described as new to science: Lucasioides digitatussp. nov., L. dissectussp. nov., L. dianensissp. nov., L. formosussp. nov., L. gracilentussp. nov., L. schmidtisp. nov. and L. subcurvatussp. nov. To date, Lucasioides species from China are 44.4% as many as all the known congeners worldwide, showing the exceptional species diversity of Lucasioides species from China. The results demonstrate that the integrative taxonomy is especially important to reveal the cryptic species among the high morphological similarity of taxa, as well as providing an effective way for species identification to accelerate the exploration of woodlice biodiversity.

 DNA barcode geometric morphometrics morphology new species Oniscidea species delimitation National Natural Science Foundation of China 501100001809 http://doi.org/10.13039/501100001809 1. Introduction

The terrestrial isopods (common name: woodlice) or Oniscidea represent a suborder within the Isopoda. More than 4,000 species in more than 500 genera and 38 families are known worldwide so far (WoRMS 2024). The suborder is a unique lineage among crustaceans completely adapted to a terrestrial way of life and occurring in almost all terrestrial habitats except for the poles and altitudes of above 4800 m (Beron 1997; Schmalfuss 2003; Schmidt and Leistikow 2004; Sfenthourakis and Taiti 2015; Campos-Filho et al. 2023). Most species of the woodlice act as keystone detritivores, and some of them are bearing importance and value in environmental monitoring and assessment, biodegradation, soil bioremediation, etc. (Bredon et al. 2020; Lebedev et al. 2020; Yang et al. 2020; Oliveira et al. 2021; Pastorino et al. 2021; Gentile et al. 2022). However, the current knowledge of the Oniscidea diversity and their dispersion ability are far from well known.

Lucasioides Kwon, 1993 is a genus of the family Agnaridae Schmidt, 2003, occurs in Oriental and Palaearctic Regions, with its distribution restricted to China, Japan, Korea and Russian Siberia (Nunomura and Xie 2000; Schmalfuss 2003; Schmidt and Leistikow 2004; Taiti and Gruber 2008; Nunomura 2013; Li 2017; Kashani 2020; Gongalsky et al. 2021; WoRMS 2024).

In the past, Lucasioides was proposed as a subgenus of Protrаcheoniscus by Arcangeli (1952), while Vandel (1969) considered it to be a full genus until Porcellio (Lucasius) gigliotosi Arcangeli, 1927 was subsequently designated as the type species (Kwon 1993). To date, thirty-one species are recorded in the genus (Nunomura 2013; WoRMS 2024), of which L. altaicus Gongalsky, Nefedief & Turbanov, 2021 represent the far northwestern range limit (Altai Mountains of southwestern Siberia, Russia) of the genus (Gongalsky et al. 2021; WoRMS 2024).

To understand the woodlice distribution, annual mean temperature has been proposed to an important limiting factor (Kuznetsova and Gongalsky 2012). As shown in Figure 1, nine Chinese Lucasioides species are recorded from the areas with the annual mean temperature ranging from 0 to 29°C. Based the conspicuously geographic distribution gaps of Chinese Lucasioides, and in where the annual mean temperature is suitable to its occurrence (Fig. 1), we deduced that potential species diversity of the genus can be revealed in further investigation.

10.3897/asp.82.e113041.figure1 AA87F62D-EFDA-5FAF-B449-C5053FAD4B69

Map of China showing the annual mean temperature (°C) and localities where Lucasioides species are recorded.

https://binary.pensoft.net/fig/1083531

At present, the ambiguous differences among some members of the genus are difficult to be verified to interspecific divergence or intraspecific variation based only in traditional morphological. Moreover, the molecular studies of the genus are scarce. In this paper, we present the first molecular data for the genus from a broad sample. DNA sequences were generated and integrated with morphological evidence to explore an effective way for species identification.

 2. Material and methods 2.1. Material

The specimens for this study were collected by tweezers, fixed in 100% ethanol, stored in the Insect Museum, Jiangxi Agricultural University, Nanchang, China (JXAUM) at –34°C.

 2.2. Methods 2.2.1. Dissections and terminology

The whole body of the specimens was placed in acid-fuchsin staining buffer for twelve hours. The appendages were dissected and mounted on micro preparations in a neutral balsam mounting medium using a Zeiss Stereo Discovery V12 microscope. The morphological terminology followed Kwon (1993) and Schmidt (2002, 2003).

 2.2.2. Images and morphological analysis

Images were taken with a camera Zeiss AxioCam Icc 5 attached to a Zeiss Stereo Discovery V12 microscope. The line drawings were drawn by the GNU Image Manipulation Program (Montesanto 2015).

For morphometric analysis, a total of 252 habitus in the dorsal view of Lucasioides species were selected. The images were converted into TPS file by tpsUtil 1.56 (Rohlf 2013). To evaluate variation in body shape, fourteen landmarks were placed on the posterior angle of each pereonite and two landmarks on the anterior protruding angle of the first pereonite. Landmarks for all images were aligned by performing Procrustes Fit, and the landmarks were recorded using TPSdig 2.17 (Rohlf 2013). To visualize shape variations across morphospace, principal component analyses (PCAs) and canonical variate analyses (CVAs) were conducted by MorphoJ (Klingenberg 2011).

 2.2.3. DNA extraction, amplification and sequencing

Genomic DNA was extracted from muscles of samples by the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit. Partial of cytochrome c oxidase subunit I (COI), and partial of 18S and 28S rRNA genes, along with a fragment of the nuclear protein-coding gene sodium-potassium ATPase α-subunit (NaK) were amplified using polymerase chain reaction (PCR). Fragment of COI gene were amplified using the primers LCO1490/HCO2198 (Folmer et al. 1994). The primers pairs 18sai/18sbi, 28sa/28sb (Whiting et al. 1997), NaK for-b/NaK rev2 (Tsang et al. 2008) were used for the amplification of 18S rRNA, 28S rRNA and NaK genes. PCR amplifications were performed with the procedure mentioned by Wang et al. (2022). PCR products were purified using 1% agarose gel electrophoresis and were sequenced by an ABI3730XL DNA Analyzer (Applied Biosystems) in Sangon Biotech Co. Shanghai, China. All sequences were deposited in DDBJ (DNA Data Bank of Japan), with accession numbers listed in Table S1. Parallel to this, voucher information, taxonomic classifications, photos, DNA barcode sequences, used primer pairs and trace files were deposited in the projects LCA and LUCAS of the Barcode of Life Data Systems (BOLD; www.boldsystems.org) (Ratnasingham and Hebert 2007). All barcodes became subject of the Barcode Index Number (BIN) system as it is implemented in BOLD, and the BINs showed in Table S1.

 2.2.4. Molecular phylogenetic analyses

The resulting forward and reverse sequences of Lucasioides members (Table S1) were assembled by SeqMan, manually checked for errors, and searched with Blast to expose contaminants. The sequences of protein-coding genes COI and NaK were aligned by MACSE (Ranwez et al. 2011), non-protein-coding genes 18S and 28S were aligned with MAFFT 7.313 (Katoh and Standley 2013), then optimized by Gblocks (Castresana 2000).

We conducted Bayesian inference (BI) and Maximum likelihood (ML) analyses by four concatenated genes (COI, 18S, 28S and NaK). The BI analyses were conducted in MrBayes 3.2.6 (Ronquist et al. 2012) on the platform of PhyloSuite (Zhang et al. 2020). Four chains of Markov chain Monte Carlo (MCMC) were run simultaneously for a total of two million. Three criteria (stable average standard deviation of the split frequencies was less than 0.01; posterior probability values tended to be stable; potential scale reduction factor was close to one) were used to determine if the Bayesian analyses had reached convergence. The sampling frequency was set to100, and the number of burn-in fraction with 0.25. We also conducted Bayesian inference (BI) based COI gene for Bayesian implementation of the Poisson tree processes model for species delimitation (bPTP) in the same process. The proportion of trees that contained the clade was given as the posterior probability (PP) on the consensus tree. The ML tree was constructed by IQ-TREE 2 with 1000 bootstrap replicates (Minh et al. 2020). ModelFinder was used during these analyses to set appropriate evolution models for each partition under the Akaike information criterion (Kalyaanamoorthy et al. 2017). In the BI analyses of the concatenated genes, GTR+F+I+G4 was selected for COI, 18S, 28S, and GTR+F+G4 was selected for NaK; in the ML analyses of the concatenated genes, TIM+F+I+G4, GTR+F+I+I+R3, TIM3+F+I+I+R2, and TIM2+F+G4 was selected for COI, 18S, 28S, and NaK. Hemilepistus klugii (Brandt, 1833), H. schirazi Lincoln, 1970, Burmoniscus kathmandius (Schmalfuss, 1983), B. mauritiensis (Taiti & Ferrara, 1983), Ligidium sichuanense Nunomura, 2002 and Ligidium acuminatum Li, 2022 were used as outgroups for phylogenetic reconstructions (accession nos. provided as Table S1). The sequences of H. klugii and H. schirazi are from Dimitriou et al. (2018). The resulting gene phylogenies were visualized in iTOL v5 (Letunic and Bork 2021).

Furthermore, pairwise p-distances of all the sequences of COI, 18S, and 28S sequences were calculated by MEGA X (Kumar et al. 2018) except for the following sequences: HK2201 (COI of HK2201 only with 396 bp, much shorter than the other sequences); NS2201, NS2203 (18S of NS2201, NS2203 only with 477 bp, much shorter than the other sequences).

In molecular species delimitations, COI was employed to generate initial species hypotheses. Automatic barcode gap discovery (ABGD) automatically clustered sequences into candidate species using online version with the relative gap width was set to one (Puillandre et al. 2012). Bayesian implementation of the Poisson tree processes model for species delimitation (bPTP) was used to assess the support for initial groupings of ABGD with default parameters (Zhang et al. 2013). The input species tree used the BI tree based on COI from the phylogenetic analyses. Following the standardized approach of DNA barcode analysis, all COI sequences of Lucasioides were also analyzed in the BIN system of BOLD (Ratnasingham and Hebert 2013).

Finally, we analysed the multilocus data (COI, 18S, 28S and NaK) under the multispecies coalescent model to delimit species with BPP 4.4 (Yang 2015). The guide tree was appointed by integrating the BI tree based on multilocus sequences and selected the “joint species delimitation and species-tree inference or unguided species delimitation (A11)” for analysis. Each analysis run twice with 100,000 generations, 0.1 burn-in fraction and sampled every five generations. Three type priors for the population size parameters (θs) and the divergence time at the root of the species tree (τ0) were assumed (Zhang et al. 2018): (i) large population sizes θ~IG (3, 0.2) and deep divergences τ0~IG (3, 0.2); (ii) large population sizes θ~IG (3, 0.2) and shallow divergences τ0~IG (3, 0.002); (iii) small population sizes θ~IG (3, 0.002) and shallow divergences τ0~IG (3, 0.002).

 2.2.5. Distribution mapping

The cartographic illustration was made using DIVA-GIS 7.5 (Hijmans et al. 2005) based on an environmental variable (annual mean temperature, Bio1) retrieved from the WorldClim database (http://www.worldclim.org) and the geographic distribution of Chinese Lucasioides species (Chen 2000, 2003; Li 2017).

 3. Results 3.1. Morphological analyses

The morphological characters of the specimens collected from China were analysed using external traits and dissected appendages. As a result, seven members were preliminarily recognized, including four known ones [L. gigliotosi (Arcangeli, 1927), L. isseli (Arcangeli, 1927), L. pedimaculatus Kwon & Taiti, 1993 and L. nudus Li, 2017], and three new species. Among them, the specimens identified as L. isseli (Arcangeli, 1927) include four kinds of phenotypes, and the specimens were recognized as L. nudus Li, 2017 have another phenotype as well. It is difficult to verify their minor differences are due to interspecific divergence or intraspecific variation, nor reveal the cryptic species based on traditional morphology.

In the results of geometric morphometrics, a total of twenty-eight principal components were obtained in the principal component analyses (PCA). The first principal component (PC1) takes up 55.15% of the total shape variation, and PC1 versus PC2 showed an overlapping distribution of species in morphospace (Fig. 2). In the canonical variate analyses (CVA), the first two canonical variables (CV1 and CV2) can classify most species (Fig. 3). Although it is difficult to separate the species with similar body shape, the CVA can make a clear difference between those species with similar mounted appendages, e.g. L. schmidtisp. nov. and L. nudus (Fig. 4). The CVA demonstrated has much better resolution than the PCA.

10.3897/asp.82.e113041.figure2 447EA916-A4B6-59FC-B3A5-403455A819B4

Scatter diagrams of geometric morphometrics with principal component analysis (PCA).

https://binary.pensoft.net/fig/1083532 10.3897/asp.82.e113041.figure3 2E7D6C22-55C4-55C2-B8F0-7C50F17FAF58

Scatter diagrams of geometric morphometrics with canonical variate analysis (CVA).

https://binary.pensoft.net/fig/1083533 10.3897/asp.82.e113041.figure4 871339C6-8642-5A04-AB8F-615DDCC2FC5C

Scatter diagrams of geometric morphometrics with canonical variate analysis (CVA) among the similar Lucasioides species.

https://binary.pensoft.net/fig/1083534 3.2. Molecular analyses

In PCR amplification, a total of 248 sequences from 66 specimens were retrieved successfully, including a mitochondrial COI gene and three nuclear genes (18S, 28S and NaK) (Table S1). We analysed the phylogenetic relationships based on the four-gene data (COI, 18S, 28S and NaK). Both Maximum likelihood (ML) and Bayesian (BI) analyses revealed eleven main clades within the genus Lucasioides [Fig. 5, maximum bootstrap values (BS) = 100, maximum Bayesian posterior probabilities (PP) = 1]. Hence, we proposed an eleven-species hypothesis based on the phylogenetic results.

10.3897/asp.82.e113041.figure5 92F359FD-2E5A-5B7E-A8FC-C3E5880C4D0A

Bayesian Consensus tree based on four-gene (COI+18S+28S+NaK) sequences. Asterisks and circles represent high node support with Bayesian posterior probabilities > 0.95 and maximum bootstrap values > 95. Coloured bars represent hypothesised species groupings.

https://binary.pensoft.net/fig/1083535

Furthermore, we calculated the pairwise p-distances based on the eleven-species hypothesis using COI, 18S, and 28S data of the Lucasioides samples. In the results of COI and 28S, the maximum intraspecific distances (COI: 0−6.54%; 28S: 0−5.34%) are much smaller than the minimum interspecific distances (COI: 10.20−21.00%; 28S: 7.82−23.70%) (Tables S2, S4), supporting the eleven-species hypothesis and the partial COI and 28S sequences can be applied as a useful DNA barcode marker. Meanwhile, the results of 18S also show the maximum intraspecific distances (0−4.38%) are smaller than the minimum interspecific distances (5.94−19.37%) except for L. subcurvatussp. nov. and L. dissectussp. nov. (Table S3). The interspecific distances between them are 4.34−13.49%, which might cause by the inconsistent lengths of the sequences between L. subcurvatus (825−1028 bp) and L. dissectus (1808−1824 bp).

In molecular species delimitation, clade boundaries provided clear limits among morphospecies. As shown in Fig. 5, the molecular delimitations (ABGD, BPP, bPTP, and BIN) generally supported the eleven-species hypothesis. However, the results also represent different hypothesised species groupings, such as, ABGD and BPP under prior ii [large population sizes θ~IG (3, 0.2) and shallow divergences τ0~IG (3, 0.002)] and prior iii [small population sizes θ~IG (3, 0.002) and shallow divergences τ0~IG(3, 0.002)] well supported the eleven-species hypothesis, but BPP under prior i [large population sizes θ~IG (3, 0.2) and deep divergences τ0~IG (3, 0.2)] supported ten-species hypothesis owing to L. dianensissp. nov. and L. subcurvatussp. nov. were delimitated as a same species; in the result of bPTP (Fig. 5), L. nudus was delimitated to three hypothesised species groupings, and the other four species (L. isseli, L. schmidtisp. nov., L. dianensissp. nov. and L. gracilentussp. nov.) were recognized to two hypothesised species groupings; in the result of BIN (Fig. 5), four species (L. nudus, L. isseli, L. dianensissp. nov. and L. gracilentussp. nov.) were recognized to two hypothesised species groupings.

Finally, we treated the eleven groups within Lucasioides as valid species by integrating the results of morphological taxonomy, geometric morphometrics, phylogenetic analyses and molecular species delimitation. Seven species are described as new to science. To date, Lucasioides species from China are 44.4% as many as all the known congeners. The subsequently described and further remarks of the new species are given in the taxonomic section.

 3.3. Taxonomy

Family Agnaridae Schmidt, 2003

Animalia Isopoda Agnaridae 93AAD4FC-3A15-5ADF-8CFE-749B42FE0372 Lucasioides Kwon, 1993 Protracheoniscus (Lucasioides) Arcangeli, 1952: 298, nomen nudum. Lucasioides Vandel, 1969: 159, nomen nudum. Lucasioides Kwon, 1993: 143. Type species.

Porcellio (Lucasius) gigliotosi Arcangeli, 1927.

 Diagnosis.

Body flat, dorsally granulated, gland pores absent. Cephalon with frontal line separated from vertex by groove, median and lateral lobes well-developed. Epimeron of first pereonite with sinuous or rounded posterior margin. Noduli laterales on pereonites 1 and 5−7 closer from lateral margins, and 2−4 shifted from lateral margins. Pereopods 1−4 with brush of long setae on sternal margins of merus and carpus. Pleopodal exopods 1−5 with Protracheoniscus-type pseudotrachea (Kwon 1993; Li 2017; Gongalsky et al. 2021).

 Animalia Isopoda Agnaridae 10F6D06C-A2BA-5F04-913D-2C6E34216005 Lucasioides dissectus https://zoobank.org/61A9C129-3A31-4DE1-981F-2BD44154438F Li & Wang sp. nov. Figures 6A , 7 Type material.

Holotype. CHINA • ♂; Jiangxi Province, Jiujiang City, Nanshan National Forest Park; 29.2514°N, 116.2071°E; el. 79 m a.s.l.; 17.viii.2022; W.C. Li leg. (DNA nos. NS2201, Prep. slide no. L22090). — Paratypes. • 3 ♂♂, 2 ♀♀; same data as the holotype (DNA nos. NS2002−NS2004); CHINA • 3 ♂♂, 5 ♀♀; Jiangxi Province, Pingxiang City, Nankeng Forest Farm; 27.4650°N, 113.8940°E, el. 590 m a.s.l.; 21.vi.2011; W.C. Li leg. (DNA nos. NKLC2001−NKLC2008, Prep. slide nos. L17321−L17326).

 Diagnosis.

Pereonite 1 with acute postero-lateral corner; pleopod 1 exopod with bilobed apex, and outer lobe approximate three times as long as inner lobe.

 Description.

Body length of males 7.0−8.5 mm, of females 7.5−9.5 mm. Color in alcohol brown, dorsum granulated, bearing irregular white muscle spots (Fig. 6A). Pereonite 1 sinuous on posterior margin of epimeron, postero-lateral corner acute. Noduli laterales as in Fig. 7A. Telson triangular, slightly wider than long, lateral margins concave, apex blunted round; uropod exopodite about twice as long as basipodite (Figs 6A, 7A). — Cephalon with triangular median lobe, median lobe shorter than lateral lobes (Figs 6A, 7A). Antennula with several aesthetascs on distal tip and antero-lateral margins of third article (Fig. 7B). Antennal flagellum with first segment two thirds as long as second one (Fig. 7C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 7D). Pereopod 7 ischium with deep depression on rostral surface, carpus slightly expanded near middle on dorsal margin (Fig. 7E). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 7F−J). — MALE: pleopod 1 exopod conspicuously bilobed at posterior tip, outer lobe approximate three times as long as inner lobe, endopod with broad basal part, narrowed towards beak-shaped posterior tip (Fig. 7F); pleopod 2 endopod styliform, nearly as long as exopod (Fig. 7G).

10.3897/asp.82.e113041.figure6 9B0D91F7-494D-5E38-A0EB-F13DB98DD711

Habitus of male Lucasioides species in dorsal view. AL. dissectussp. nov.; BL. isseli (Arcangeli, 1927); CL. dianensissp. nov.; DL. digitatussp. nov.; EL. schmidtisp. nov.; FL. nudus Li, 2017; GL. formosussp. nov.; HL. gracilentussp. nov.; IL. subcurvatussp. nov. (Scale: 1 mm).

https://binary.pensoft.net/fig/1083536 10.3897/asp.82.e113041.figure7 93CCB7E4-5940-56E3-B71F-64B44219ED61

Lucasioides dissectussp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083537 Etymology.

Latin “dissectus” = partite. The new species name refers to the male pleopod 1 exopod conspicuously bilobed at apical apex.

 Remarks.

The new species is very similar to L. isseli (Arcangeli, 1927) in having the apical tip of pleopod 1 exopod conspicuously bilobed, and the outer lobe much longer than the inner lobe. But it can be distinguished by the shape of median lobe of the cephalon angled in anterior middle margin, and pereonite 1 with an acute postero-lateral corner (Fig. 6A). In L. isseli, the median lobe of the cephalon is arched in anterior middle margin, and pereonite 1 with a broad rounded postero-lateral corner (Fig. 6B). This species also resembles L. dianensissp. nov. in having the apical tip of pleopod 1 exopod conspicuously bilobed (Figs 6A, 7 versus 6C, 8). But it can be distinguished in having the outer lobe of pleopod 1 exopod three times as long as inner lobe (Fig. 7F). In L. dissectus, the outer lobe is twice as long as inner lobe (Fig. 8F). However, the above three species with minor differences in the external appearances or mounted appendages. We are not sure these ambiguous different traits belong to intraspecific variations or interspecific divergences. In the morpho-geometric analysis of geometric morphometrics, the scatter diagrams indicate that the CVAs were able to clearly classify the three species with the first two canonical variables (Figs 3, 4). Furthermore, they can also be separated based on molecular analyses (Fig. 5). Thus, we clarified the above dilemma by using the integrative approaches.

 Animalia Isopoda Agnaridae 3003F925-53DA-5B5A-8B56-37F361BE2962 Lucasioides dianensis https://zoobank.org/E1E83038-A6A6-4A8F-B9DA-7B7A486A8C67 Li & Wang sp. nov. Figures 6C , 8 Type material.

Holotype. CHINA•1♂; Yunnan Province, Yuxi City, Tonghai County, Xiushan Historical and Cultural Park; 24.0978°N, 102.7446°E, el. 1989 m a.s.l; 2.viii.2022; J. Wang, X.K. Hong leg. (DNA nos. XS2201, Prep. slide no. L22077). — Paratypes. • 2♂♂, 3♀♀; same data as the holotype (DNA nos. XS2202−XS2206). CHINA • 2♂♂; Yunnan Province, Kunming City, Xishan Park, Taihuasi; 26.9615°N, 102.6303°E; el. 2149 m a.s.l.; 2.viii.2021; J. Wang, X.G. Zeng, Z.L. Wan leg. (DNA nos. THS2101, THS2105, Prep. slide no. L21053).

 Diagnosis.

Pleopod 1 exopod with bilobed apex, and outer lobe twice as long as inner lobe, each lobe with one single spine.

 Description.

Body length of males 6.0−9.8 mm, of females 7.0−9.5 mm. Color in alcohol blackish brown, dorsum granulated, bearing irregular white muscle spots (Fig. 6C). Pereonite 1 sinuous on posterior margin of epimeron, postero-lateral corner acute. Noduli laterales as in Fig. 8A. Telson triangular, posterior half conspicuously narrowed towards blunted apex, lateral margin concave; uropod exopodite about twice as long as basipodite (Figs 6C, 8A). — Cephalon with triangular median lobe, median lobe loner than lateral lobes (Figs 6C, 8A). Antennula with several aesthetascs on distal tip of third article (most aesthetascs lost before dissection) (Fig. 8B). Antennal flagellum with second segment nearly twice as long as first one (Fig. 8C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 8D). Pereopod 7 ischium with deep depression on rostral surface, carpus expanded on dorsal margin (Fig. 8E). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 8F−J). — MALE: pleopod 1 exopod bilobed at posterior tip, outer lobe twice as long as inner lobe, and each lobe with spine, endopod with broad basal part, narrowed towards apex, posterior tip beak-shaped and bent outwards (Fig. 8F); pleopod 2 exopod with a line of setae on posterior part of outer margin, endopod styliform, nearly as long as exopod (Fig. 8G).

10.3897/asp.82.e113041.figure8 2CE05659-E073-5120-830B-BBF6E97B0906

Lucasioides dianensissp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083538 Etymology.

The new species is named after its type locality Yunnan Province, and “dian” is the shortened form of this province of China.

 Remarks.

The new species is very similar to L. isseli (Arcangeli, 1927) in overall appearance and male pleopods, but it can be distinguished in having two setae at the distal apex of the male pleopod 1 exopod. Furthermore, the molecular data recognized both species as distinct taxonomic entities they can also be separated based on molecular analyses (Fig. 5).

 Animalia Isopoda Agnaridae 9E03525F-6406-5ADB-AC48-828858450AF9 Lucasioides digitatus https://zoobank.org/A6901C4F-B84F-466A-9D22-DF698540BBF7 Li & Wang sp. nov. Figures 6D , 9 Type material.

Holotype. CHINA • 1 ♂; Jiangxi Province, Yichun City, Jingan County, Sanzhualun; 29.0490°N, 115.2611°E, el. 220 m a.s.l.; 19.vii.2012; W.C. Li leg. (DNA no. SZL2002, Prep. slide nos. L17309−L17314). — Paratypes. • 3♀♀; same data as the holotype (DNA nos. SZL2001, SZL2003 and SZL2004).

 Diagnosis.

Cephalon with arched median lobe; pereonite 1 with blunted postero-lateral corner; pleopod 1 exopod with finger-like outer lobe, inner lobe inconspicuous.

 Description.

Body length of males 6.2 mm, of females 6.3−8.0 mm. Color in alcohol blackish brown or yellowish brown, dorsum granulated, bearing irregular white muscle spots (Fig. 6D). Pereonite 1 sinuous on posterior margin of epimeron, postero-lateral corner blunted. Noduli laterales on pereonites as in Fig. 9A. Telson triangular, width about one and half times as long as length, lateral margin slightly concave, apex blunted round; uropod exopodite about twice as long as basipodite (Figs 6D, 9A). — Cephalon with arched median lobe, median lobe nearly as long as lateral lobes (Figs 6D, 9A). Antennula with several aesthetascs on distal tip and antero-lateral margins of third article (Fig. 9B). Antennal flagellum with second segment twice as long as first one (Fig. 9C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 9D). Pereopod 7 ischium with deep depression on rostral surface, carpus slightly expanded near middle on dorsal margin (Fig. 9E). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 9F−J). — MALE: pleopod 1 exopod slightly concave at posterior margin, outer side with finger-like lobe, inner lobe inconspicuous, endopod with broad basal part, narrowed towards beak-shaped posterior tip (Fig. 9F); pleopod 2 endopod styliform, nearly as long as exopod (Fig. 9G).

10.3897/asp.82.e113041.figure9 F2E1620F-6238-536F-B6F0-A32B984A5AD2

Lucasioides digitatussp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083539 Etymology.

Latin “digitatus” = finger-like. The new species name refers to the male pleopod 1 exopod with a finger-like lobe on outer side.

 Remarks.

The new species is very similar to L. isseli (Arcangeli, 1927) in having the cephalon with an arched median lobe, and the first pereonite with a blunted postero-lateral corner. But it can be distinguished in having the pleopod 1 exopod with an undeveloped inner lobe (Fig. 9F). In L. isseli, the inner lobe of pleopod 1 exopod is well-developed (Kwon 1993: fig. 168). In the mounted appendages, this species resembles L. nudus Li, 2017 and L. schmidtisp. nov. Furthermore, the molecular data recognized both species as distinct taxonomic entities (Fig. 5).

 Animalia Isopoda Agnaridae 0F36BBF9-7203-5C5C-A437-02DB22361F18 Lucasioides schmidti https://zoobank.org/0ADFE211-A776-4278-B034-9190DEE14FC7 Li & Wang sp. nov. Figures 6E , 10 Type material.

Holotype. CHINA • 1 ♂; Hunan Province, Huaihua City, Xupu County, Lingcui Mountain Park; 27.9074°N, 110.5778°E, el. 214 m a.s.l.; 15.viii.2019; W.C. Li, J.B. Yang leg. (DNA no. LCS2020, Prep. slide nos. L2046−L2048). — Paratypes. • 2 ♀♀; same data as the holotype (DNA no. LCS2008). CHINA • 1 ♂, Hunan Province, Loudi City, Xinhua County, Shizi Mountain Park; 27.7311°N, 110.3338°E, el. 235 m a.s.l.; 16.vii.2019; W.C. Li, J.B. Yang leg. (DNA no. SZS1901, Prep. slide nos. L19034−L19036).

 Diagnosis.

Pereonite 1 slightly sinuous on posterior margin of epimeron, postero-lateral corner nearly right-angled; pleopods 1, 3−5 exopods out curved on outer margins; pleopod 1 exopod with outer lobe much longer than inner lobe.

 Description.

Body length of males 4.6−6.9 mm, of females 5.51 mm. Color in alcohol blackish brown, dorsum slightly granulated, bearing irregular white muscle spots. Pereonite 1 slightly sinuous on posterior margin of epimeron, postero-lateral corner nearly right-angled (Fig. 6E). Noduli laterales as in Fig. 10A. Telson triangular, length about two thirds as long as width, lateral margin concave, apex blunted round; uropod exopodite about two and half as long as basipodite (Figs 6E, 10A). — Cephalon with arched median lobe, median lobe slightly longer than lateral lobes (Figs 6E, 10A). Antennula with several aesthetascs on distal tip of third article (Fig. 10B). Antennal flagellum with first segment about third as long as second one (Fig. 10C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 10D). Pereopod 7 ischium with deep depression on rostral surface, carpus slightly expanded on dorsal margin (Fig. 10E). — Pleopods 1−5 exopod with Protracheoniscus-type pseudotrachea (Fig. 10F−J). — MALE: pleopod 1 exopod bilobed at posterior tip, outer lobe much longer than inner lobe, endopod with broad basal part, narrowed towards beak-shaped posterior tip (Fig. 10F); pleopod 2 endopod styliform, a bit longer than exopod (Fig. 10G).

10.3897/asp.82.e113041.figure10 D4DC1B9C-7B32-5498-84F5-57E1F00CEFC4

Lucasioides schmidtisp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083540 Etymology.

The new species is named after Dr. Christian Schmidt (Senckenberg Naturhistorische Sammlungen Dresden, Germany), who contributed profoundly to systematic research in woodlice; noun (name) in the genitive case.

 Remarks.

The new species is very similar to L. nudus Li, 2017 in having the outer lobe of the pleopod 1 exopod much longer than the inner lobe. But it can be distinguished by the slenderer body shape and less developed epimeron (Fig. 6E versus 6F). Furthermore, the molecular data recognized both species as distinct taxonomic entities (Fig. 5).

 Animalia Isopoda Agnaridae 9B6AFA91-B424-5D41-9EB1-3B4232A47FFA Lucasioides formosus https://zoobank.org/989DCD87-7808-4D8F-B7AD-49FA36C7A95C Li & Wang sp. nov. Figures 6G , 11 Type material.

Holotype. CHINA • 1♂; Yunnan Province, Lijiang City, Yulong County, Liming Lisu Ethnic Township, Houka; 27.1469°N, 99.8193°E, el. 1921 m a.s.l.; 8.vii.2022; J. Wang, X.K. Hong leg. (DNA no. HK2201, Prep. slide no. L22081). — Paratypes. • 2 ♂♂, 2 ♀♀; same data as the holotype (DNA nos. HK2202−HK2205). CHINA • 1 ♂, 4 ♀♀; Yunnan Province, Lijiang City, Zimei lake; 26.9891°N, 100.1966°E, el. 2724 m a.s.l.; 7.vii.2022; J. Wang, X.K. Hong leg. (DNA nos. ZMH2201−ZMH2205, Prep. slide no. L22082). • 2 ♂♂, 4 ♀♀; Yunnan Province, Dali City, Jianchuan County, Qianshi Mountain; 26.5328°N, 99.8877°E, el. 2364 m a.s.l.; 11.vii.2022; J. Wang, X.K. Hong leg. (DNA nos. QSS2201−QSS2206, Prep. slide no. L22076). • 4♀♀; Yunnan Province, Lijiang City, Gucheng District; 26.9258°N, 100.2079°E, el. 2465 m a.s.l.; 7.vii. 2022; J. Wang, X.K. Hong leg. (DNA nos. LJGC2201−LJGC2204). • 3♀♀; Yunnan Province, Lijiang City, Meiquan Village; 26.9058°N, 100.1499°E, el. 2486 m a.s.l.; 6.vii.2022; J. Wang, X.K. Hong leg. (DNA nos. MQC2201−MQC22032205).

 Diagnosis.

Pereonite 1 slightly sinuous on posterior margin of epimeron, postero-lateral corner nearly right-angled; noduli laterales on pereonites 1−4 shifted from lateral margins than those of pereonites 5−7; pereopod 7 remarkably expanded on dorsal margin of carpus; pleopod 1 exopod slightly concave at posterior tip.

 Description.

Body length of males 6.3−8.0 mm, of females 5.5−11.0 mm. Color in alcohol blackish brown, dorsum granulated, bearing irregular yellowish white muscle spots (living individuals with yellowish blue muscle spots). Pereonite 1 slightly sinuous on posterior margin of epimeron, postero-lateral corner nearly right-angled (Fig. 6G). Noduli laterales on pereonites 1−4 shifted from lateral margins than those of pereonites 5−7 (Fig. 11A). Telson triangular, width about twice as long as length, lateral margin slightly concave, apex blunted; uropod exopodite about three times as long as basipodite (Figs 6G, 11A). — Cephalon with arched median lobe, median lobe longer than lateral lobes (Figs 6G, 11A). Antennula with several aesthetascs on distal tip of third article (Fig. 11B). Antennal flagellum with second segment twice as long as first one (Fig. 11C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 11D). Pereopod 7 ischium without deep depression on rostral surface, carpus remarkably expanded near middle on dorsal margin (Fig. 11E). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 11F−J). — MALE: pleopods 1 exopod slightly concave at posterior tip, forming two inconspicuous lobes, endopod with broad basal part, narrowed towards apex, posterior tip beak-shaped and bent outwards (Fig. 11F); pleopod 2 exopoddistinctive concave on outer margin, posterior two fifths with several setae, endopod styliform, slightly longer than exopod (Fig. 11G).

10.3897/asp.82.e113041.figure11 DB64C7A7-8AED-55AC-9578-16ECEFB2BA68

Lucasioides formosussp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083541 Etymology.

Latin “formosus” = beautiful. The new species name is an allusion to beautiful yellowish blue muscle spots on the dorsum of the living individuals.

 Remarks.

The new species is not strictly consistent with the generic characters of Lucasioides because of the noduli laterales on pereonites 1−4 shifted from lateral margins than those of pereonites 5−7 instead of the noduli laterales on pereonites 2−4 much farther from lateral margins than those of pereonites 1, 5−7. It is difficult to give a ready answer for this dilemma based on the morphological evidence alone. In the phylogenetic analyses, the relationship between this species and the other Lucasioides species is well-supported (Fig. 5). Hence, we assigned this species as a member of Lucasioides by integrating molecular and morphological evidence in this context.

 Animalia Isopoda Agnaridae 50411D4E-0E55-5891-9BE3-DA4F1F44577A Lucasioides gracilentus https://zoobank.org/AF1F4B68-8711-40C1-8842-062D403B46A5 Li & Wang sp. nov. Figures 6H , 12 Type material.

Holotype. CHINA • 1 ♂; Fujian Province, Putian City, Hugong Mountain; 25.3490°N, 119.0077°E, el. 462 m a.s.l.; 4.vii.2021; X.G. Zeng, J. Wang leg. (DNA no. HGS2104, Prep. slide no. L21060). — Paratypes. • 5 ♂♂, 3 ♀♀, same data as the holotype (DNA nos. HGS2105−HGS2112).

 Diagnosis.

Pereonite 1 nearly straight on posterior margin of epimeron, postero-lateral corner convex; pleopod 1 exopod concave at posterior apex and formed two broad lobes, inner lobe longer and broader than outer lobe.

 Description.

Body length of males 4.8−5.0 mm, of females 6.0−8.0 mm. Color in alcohol blackish brown, dorsum granulated, bearing irregular white muscle spots (Fig. 6H). Pereonite 1 nearly straight on posterior margin of epimeron, postero-lateral corner convex. Noduli laterales on pereonites 2−4 farthest from lateral margin, noduli laterales on pereonites 1 and 7 slightly closer to lateral margin, and noduli laterales on pereonites 5−6 closest to lateral margin (Fig. 12A). Telson triangular, width about twice as long as length, lateral margin slightly concave, apex blunted; uropod exopodite about two and half as long as basipodite (Figs 6H and 12A). — Cephalon with arched median lobe, median lobe much longer than lateral lobes (Figs 6H and 12A). Antennula with several aesthetascs on distal tip of third article. Antennal flagellum with second segment two and half as long as first one (Fig. 12B). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 12C). Pereopod 7 ischium without deep depression on rostral surface, carpus unexpanded on dorsal margin (Fig. 12D). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 12E−I). — MALE: pleopod 1 exopod concave at posterior tip and formed two broad lobes, inner lobe longer and broader than outer lobe, endopod with broad basal part, narrowed towards beak-shaped posterior tip (Fig. 12E); pleopod 2 endopod styliform, nearly as long as exopodite (Fig. 12F).

10.3897/asp.82.e113041.figure12 49BBD8D4-0EB0-5F42-A31B-B3F38388D805

Lucasioides gracilentussp. nov. A Holotype in dorsal view; B antenna; C pereopod 1; D pereopod 7; E pleopod 1; F pleopod 2; G pleopod 3 exopod; H pleopod 4 exopod; I pleopod 5 exopod.

https://binary.pensoft.net/fig/1083542 Etymology.

Latin “gracilentus” = slender. The new species name refers to the slender habitus of the specimens.

 Remarks.

The new species is also inconsistent within Lucasioides if we recognized it by using the morphological traits alone. However, all the mounted structures of this species support it to be assigned as Lucasioides member. Furthermore, the phylogenetic relationship between this species and the other Lucasioides species is well-supported (Fig. 5).

The new species is similar to L. boninshimensis (Nunomura, 1987) in having the convex postero-lateral corner of pereonite 1. But it can be distinguished by the pleopod 1 exopod with inner lobe broader than outer lobe, endopod with a beak-shaped posterior tip (Fig. 12E). In L. boninshimensis, the pleopod 1 exopod with inner as wide as outer lobe, endopod with a straight posterior tip (Nunomura 1987: fig. 115I).

 Animalia Isopoda Agnaridae 15D7C52A-0685-5BD7-A1D5-108A33FEF84F Lucasioides subcurvatus Li & Wang sp. nov. Figures 6I , 13 Type material.

Holotype. CHINA • 1 ♂; Sichuan Province, Leshan City, Mabian Yi Autonomous County, Minjian Town, Zhang Youfang Village; 28.8439°N, 103.5475°E, el. 600 m a.s.l.; 12.viii. 2012; W.C. Li leg. (DNA no. DFD2006, Prep. slide nos. L2073−L2075). — Paratypes. • 1 ♂, 3 ♀♀; same data as the holotype (DNA nos. DFD2003b, DFD2003−DFD2005). CHINA • 1 ♂, Guizhou Province, Bijie City, Qianxi County, Qianxi Railway Station; 26.9937°N, 106.0413°E, el. 1221 m a.s.l.; 4.viii. 2020; X.G. Zeng, J.B. Yang leg. (DNA no. GQX2204, Prep. slide no. L22098).

 Diagnosis.

Pereonite 1 with right-angled postero-lateral corner; pleopod 1 exopod slightly concave, forming two inconspicuous lobes.

 Description.

Body length of males 6.1−6.9 mm, of females 5.2−6.0 mm. Color in alcohol blackish brown, dorsum slightly granulated, and with irregular white muscle spots (Fig. 6I). Pereonite 1 slightly sinuous on posterior margin of epimeron, postero-lateral corner nearly right-angled. Noduli laterales as in Fig. 13A. Telson triangular, width almost as long as length, lateral margin slightly concave, posterior apex blunted; uropod exopodite about two and half times as long as basipodite (Figs 6I, 13A). — Cephalon with arched median lobe, median lobe nearly as long as lateral lobes (Figs 6I and 13A). Antennula with several aesthetascs on distal tip of third article (Fig. 13B). Antennal flagellum with first segment about three fifths as long as second one (Fig. 13C). — Pereopod 1 with brush of long setae on carpus and merus (Fig. 13D). Pereopod 7 ischium with deep depression on rostral surface, carpus slightly expanded near middle on dorsal margin (Fig. 13E). — Pleopods 1−5 exopods with Protracheoniscus-type pseudotrachea (Fig. 13F−J). — MALE: pleopod 1 exopod slightly concave at posterior tip, forming two inconspicuous lobes, endopod with broad basal part, narrowed towards beak-shaped posterior tip (Fig. 13F); pleopod 2 endopod styliform, a bit longer than exopod (Fig. 13G).

10.3897/asp.82.e113041.figure13 43F1A04F-EBC6-56E7-947E-6CC64987F8F6

Lucasioides subcurvatussp. nov. A Holotype in dorsal view; B antennula; C antenna; D pereopod 1; E pereopod 7; F pleopod 1; G pleopod 2; H pleopod 3 exopod; I pleopod 4 exopod; J pleopod 5 exopod.

https://binary.pensoft.net/fig/1083543 Etymology.

Latin “subcurvatus” = subcurved. The new species name refers to the shape of pleopod 1 exopod with a subcurved apex.

 Remarks.

The new species is very similar to L. formosussp. nov. in having the pleopod 1 exopod slightly concave and forming two inconspicuous lobes. But it is easy to be distinguished in having the noduli laterales on pereonites 2−4 shifted from lateral margins than the noduli laterales on pereonites 1, 5−7 (Fig. 13A). In L. formosus, the noduli laterales on pereonites 1−4 are much farther from lateral margin than noduli laterales of pereonites 5−7 (Fig. 11A). Furthermore, the two species can also be separated based on morphometric evidence and molecular analyses (Figs 3, 5).

 3.4. Key to the species of the genus <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Lucasioides">Lucasioides</tp:taxon-name-part></tp:taxon-name></italic>
1 Pereonite 1 concave near lateral side of posterior margin 2
1’ Pereonite 1 not concave near lateral side of posterior margin 32
2 Pereonite 1 slightly concave near lateral side of posterior margin 3
2’ Pereonite 1 obviously sinuate near lateral side of posterior margin 14
3 Male pleopod 1 exopod not concave at distal apex 4
3’ Male pleopod 1 exopod concave at distal apex 7
4 Male pleopod 1 exopod half-moon-shaped 5
4’ PMale pleopod 1 exopod triangular or bean-shaped 6
5 Male pleopod 2 exopod conspicuously concave near middle of outer margin (Nunomura 1991: fig. 169) L. nakadoriensis (Nunomura, 1991)
5’ Male pleopod 2 exopod nearly straight in middle of outer margin (Nunomura 1987: fig. 107; Nunomura 2010a: figs. 7) L. nishimurai (Nunomura, 1987)
6 Male pleopods 1−2 exopods triangular (Nunomura and Xie 2000: fig. 11) L. daliensis Nunomura & Xie, 2000
6’ Male pleopod 1 exopod bean-shaped, male pleopod 2 exopod broad at basal part, distal two thirds thin and long (Nunomura and Xie 2000: fig. 12) L. longicaudatus Nunomura & Xie, 2000
7 Male pleopod 1 exopod with inner lobe as long as outer lobe 8
7’ Male pleopod 1 exopod with inner lobe not as long as outer lobe 10
8 Noduli laterales on pereonite 7 much farther from lateral margins (Kwon and Taiti 1993: fig. 188) L. cavernicolus Kwon & Taiti, 1993
8’ Noduli laterales on pereonite 7 close to lateral margins 9
9 Noduli laterales on pereonite 1 close to lateral margins (Fig. 13) L. subcurvatus Li & Wang sp. nov.
9’ Noduli laterales on pereonite 1 much farther from lateral margins (Fig. 11) L. formosus Li & Wang sp. nov.
10 Male pleopod 1 exopod with inner lobe longer than outer lobe 11
10’ Male pleopod 1 exopod with inner lobe shorter than outer lobe 13
11 Male pleopod 1 exopod conspicuously concave at distal apex (Arcangeli 1927: fig. 12) L. zavattarii (Arcangeli, 1927)
11’ Male pleopod 1 exopod nearly straight at distal apex 12
12 Antenna flagellum with second segment about twice as long as first one; inner lobe of male pleopod 1 exopod weakly developed (Nunomura and Xie 2000: fig. 13) L. xiaoi Nunomura & Xie, 2000
12’ Antenna flagellum with second segment about three time as long as first one; inner lobe of male pleopod 1 exopod well developed (Nunomura 1987: fig. 105) L. minatoi (Nunomura, 1987)
13 Epimeron well developed; postero-lateral corner of pereonite 1 acute (Li 2017: fig. 2) L. nudus Li, 2017
13’ Epimeron undeveloped; postero-lateral corner of pereonite 1 nearly right-angled (Fig. 10) L. schmidti Li & Wang sp. nov.
14 Male pleopod 1 exopod concave at distal apex 15
14’ Male pleopod 1 exopod not concave at distal apex 26
15 Distal margin of male pleopod 1 exopod truncated (Nunomura 2013b: fig. 1) L. kurehaensis Nunomura, 2013
15’ Distal margin of male pleopod 1 exopod untruncated 16
16 Male pleopod 1 exopod with inconspicuous outer lobe 17
16’ Male pleopod 1 exopod with clear outer lobe 18
17 Apical part of male pleopod 2 exopod short and broad (Nunomura 2000: fig. 4) L tokyoensis Nunomura, 2000
17’ Apical part of male pleopod 2 exopod thin and long (Nunomura 2008: fig. 1) L. toyamaensis Nunomura, 2008
18 ale pleopod 1 exopod with inner lobe as long as outer lobe (Nunomura 2013a: 27, fig. 4) L. albulus Nunomura, 2013
18’ Male pleopod 1 exopod with inner lobe not as long as outer lobe 19
19 Male pleopod 1 exopod inner lobe longer than outer lobe 20
19’ Male pleopod 1 exopod inner lobe shorter than outer lobe 21
20 Outer lobe of male pleopod 1 exopod with sinuate margin (Nunomura 2008: fig. 4) L. sagarai Nunomura, 2008
20’ Outer lobe of male pleopod 1 exopod with smooth margin (Kwon 1993: fig. 8) L. gigliotosi (Arcangeli, 1927)
21 Outer lobe of male pleopod 1 exopod with sinuate margin (Nunomura 2010c: fig. 1) L. yokohatai Nunomura, 2010
21’ Outer lobe of male pleopod 1 exopod with smooth margin 22
22 Male pleopod 1 exopod with inner lobe shorter than outer lobe 23
22’ Male pleopod 1 exopod with inner lobe much shorter than outer lobe 24
23 Male pleopod 1 exopod with inner lobe narrower than outer lobe, inner lobe ending with seta (Nunomura 1987: fig. 104; Kwon 1995: fig. 9) L. sinuosus (Nunomura, 1987)
23’ Male pleopod 1 exopod inner lobe wider than outer lobe, each lobe ending with seta (Fig. 8) L. dianensis Li & Wang sp. nov.
24 Male pleopod 1 exopod with well developed inner lobe (Fig. 7) L. dissectus Li & Wang sp. nov.
24’ Male pleopod 1 exopod with weakly developed inner lobe 25
25 Male pleopod 1 exopod with inner lobe much wider than outer lobe (Fig. 9) L. digitatus Li & Wang sp. nov.
25’ Male pleopod 1 exopod with inner lobe as wide as outer lobe (Kwon and Taiti, 1993: fig. 168) L. isseli (Arcangeli, 1927)
26 Male pleopod 1 exopod triangular 27
26’ Male pleopod 1 exopod not triangular 28
27 Male pleopod 1 exopod triangular, concave near middle (Nunomura 2013a: fig. 2) L. yamamotoi Nunomura, 2013
27’ Male pleopod 1 exopod triangular, not concave near middle (Uljanin 1875: fig. 7) L. latus (Uljanin, 1875)
28 Male pleopod 1 exopod ovate 29
28’ Male pleopod 1 exopod half-moon-shaped 30
29 Inner margin of male pleopod 1 exopod slightly concave near middle (Nunomura 1987: fig. 110) L. sakimori (Nunomura, 1987)
29’ Inner margin of male pleopod 1 exopod not concave near middle (Nunomura 1987: fig. 102) L. kobarii (Nunomura 1987)
30 Outer margin of male pleopod 1 exopod distinctively concave near middle (Nunomura 2010b: fig. 3) L. ashiuensis Nunomura, 2010
30’ Outer margin of male pleopod 1 exopod not concave near middle 31
31 Noduli laterales on pereonites almost at same distance from lateral margins; male pleopod 1 exopod with seta at distal apex (Nunomura 2003b: fig. 7) L. nichinanensis Nunomura, 2003
31’ Noduli laterales on pereonites 2–4 shifted from lateral margins than those of pereonites 5−7; male pleopod 1 exopod without seta at distal apex (Nunomura 1987: fig. 108) L. hachijoensis (Nunomura, 1987)
32 Posterior lateral margin of pereonite 1 nearly straight 33
32’ Posterior lateral margin of pereonite 1 convex 35
33 Male pleopod 1 exopod slightly concave at distal apex (Kwon and Taiti 1993: fig. 185) L. pedimaculatus Kwon & Taiti, 1993
33’ Male pleopod 1 exopod concave at distal apex 34
34 Noduli laterales on pereonites 2–4 shifted from lateral margins than those of pereonites 5−7; male pleopod 1 exopod with inner lobe as long as outer lobe (Kwon 1993: figs. 9–10) L. taitii Kwon, 1993
34’ Noduli laterales on pereonites almost at same distance from lateral margin; male pleopod 1 exopod with inner lobe shorter than outer lobe (Gongalsky et al. 2021: 142, figs. 2, 6) L. altaicus Gongalsky, Nefediev & Turbanov, 2021
35 Male pleopod 1 exopod concave at distal apex 36
35’ Male pleopod 1 exopod not concave at distal apex 37
36 Noduli laterales on pereonites almost at same distance from lateral margins; male pleopod 1 exopod with seta at distal apex of inner lobe (Nunomura 1987: fig. 115) L. boninshimensis (Nunomura, 1987)
36’ Noduli laterales on pereonites 2–4 shifted from lateral margins than those of pereonites 5−7; male pleopod 1 exopod with seta at distal apex of outer lobe (Fig. 12) L. gracilentus Li & Wang sp. nov.
37 Male pleopod 1 exopod with round distal apex (Nunomura 2013a: fig. 6) L. punctatus Nunomura, 2013
37’ Male pleopod 1 exopod with triangular distal apex (Nunomura 2003a: fig. 4) L. minakatai Nunomura, 2003
4. Discussion

To date, thirty-eight species are recorded in the genus Lucasioides all over the world, including the new species described herein. As mentioned, all the recorded localities of the genus are from China, Japan, Korea, and Russian Siberia (Nunomura and Xie 2000; Schmalfuss 2003; Schmidt and Leistikow 2004; Taiti and Gruber 2008; Nunomura 2013; Li 2017; Kashani 2020; Gongalsky et al. 2021; WoRMS 2024). Among them, L. altaicus Gongalsky, Nefedief & Turbanov, 2021 occurs in the northwestern range limit (Russian Siberia), while L. zavattarii (Arcangeli, 1927) in the southeastern range limit (Chinese Hong Kong) (Kwon and Taiti 1993; Gongalsky et al. 2021; WoRMS 2024). Based on our field experience, specimens of Lucasioides usually live in the leaf litters or under the rock stacks in most biomes without strictly climatic requirements. It means that the genus has many suitable habitats in the presently geographic distribution gaps between the sub-tropical zone and sub-frigid zone. Hence, the Asian fauna may hide higher diversity of Lucasioides species.

The effective taxonomic approaches are essential to explore the species diversity of taxa. To solve the morphological problems of woodlice, DNA-based approach has revealed an effective way for delimiting species boundaries and revealing cryptic taxa (e.g., Karasawa et al. 2014; Karasawa 2016; Bedek et al. 2019; Zeng et al. 2021; Khalaji-Pirbalouty et al. 2022; Raupach et al. 2022; Wang et al. 2022a; Yoshino and Kubota 2022). However, there are various factors affect the accuracy of molecular species delimitations. The hybridization and phylogeographic effects have been observed in several woodlice (Bilton et al. 1999; Gentile et al. 2010; Eberl 2013; Hurtado et al. 2014; Lee et al. 2014), and the atypical mitochondrial genome composed of linear monomes or circular dimes may also lead to mitochondrial gene heterogeneity, casting a shadow over the DNA barcode (Chandler et al., 2015; Marcadé et al., 2007). In addition to these factors, the co-amplification of nuclear mitochondrial pseudogenes may lead to misestimate species diversity (Song et al. 2008; Buhay 2009). The above factors or various combinations of these factors presumably cause the high intraspecific variability within the DNA barcode fragments (Raupach et al. 2022). Thus, it is essential to apply an integrative approach to objectively evaluate the threshold value of intraspecific and interspecific genetic variations in various genera of the suborder Oniscidea.

As presented here, the first molecular analyses based on a broad sample of Lucasioides (Table S1), supporting the partial COI sequences can be applied as a useful DNA barcode marker for identifying Lucasioides species (Table S2). In molecular species delimitation, ABGD and BPP strongly supported a same species hypothesis, but bPTP and BIN may overestimate the species diversity of the genus (Fig. 5). These results show that the different model may solve the problem of uncertainty and make evaluation results more reliable and precise.

In addition, we applied geometric morphometrics to visualize and test the body-shaped differences among the Lucasioides samples using two-D landmarks data. Results demonstrated that canonical variate analysis is superior to principal component analysis (Fig. 2 versus 3) in exploring morphological differences in the genus. This could be attributed to the properties of the different multivariate statistical techniques between PCA and CVA (Heymann and Noble 1989; Wang et al. 2022). Despite the geometric morphometrics cannot absolutely delimit the high morphological similarity of Lucasioides species, it can provide helpful morphometric evidence to separate them.

Finally, we integrated DNA sequences with morphological evidence to resolve the taxonomic problems, revealing exceptional species diversity of Lucasioides from China and four cryptic species. The results demonstrate that the integrative taxonomy is especially important to reveal the cryptic species among the high morphological similarity of taxa, as well as providing an effective way for species delimitation to accelerate the exploration of woodlice biodiversity.

 5. Authors’ contributions

JW, CJ and WCL contributed to the study conceptualization and design. JW, CHY and WCL identified specimens and produced illustrations and maps. JW carried out the molecular laboratory work under supervision of WCL and CJ. All authors contributed to the draft of the manuscript. All authors read and approved the final manuscript.

 6. Acknowledgements

We are grateful to Dr. S. Taiti (Istituto per lo Studio degli Ecosistemi, Italy), Dr. C. Schmidt (Senckenberg Naturhistorische Sammlungen Dresden, Germany), Dr. N. Nunomura (Institute of Nature and Environmental Technology, Kanazawa University, Japan), Dr. D. H. Kwon (Inje University, Korea), and Dr. G. M. Kashani (University of Zanjan, Iran) for providing important references and generous help. Special thanks are given to Dr. M. J. Raupach (Bavarian State Collection of Zoology, Germany) and an anonymous reviewer for their insightful suggestions. The research was supported by the National Natural Science Foundation of China (nos. 31960100, 82073972).

 7. References ArcangeliA (1927) Isopodi terrestri raccolti nell’Estreme Oriente dal Prof. Filippo Silvestri. Bollettino del Laboratorio di Zoologia Generale e Agraria del R. Istituto Superiore Agrario in Portici 20, 211–269. ArcangeliA (1952) Correzioni riguardanti crostacei isopodi terrestri dell’Estremo Oriente. Archivio Zoologico Italiano 37: 291−326. BedekJTaitiSBilandžijaHRistoriEBarattiM (2019) Molecular and taxonomic analyses in troglobiotic Alpioniscus (Illyrionethes) species from the Dinaric Karst (Isopoda: Trichoniscidae).Zoological Journal of the Linnean Society187: 539–584. BeronP (1997) On the high mountain IsopodaOniscidea in the Old World.Historia naturalis bulgarica8: 85–100. BiltonDTGoodeDMalletJ (1999) Genetic differentiation and natural hybridization between two morphological forms of the common woodlouse, Oniscus asellus Linnaeus 1758.Heredity82: 462–469. https://doi.org/10.1038/sj.hdy.6885170 BredonMHerranBBertauxJGrèvePMoumenBBouchonD (2020) Isopod holobionts as promising models for lignocellulose degradation. Biotechnol Biofuels 13: 49. BuhayJE (2009) “COI-like” sequences are becoming problematic in molecular systematic and DNA barcoding studies.Journal of Crustacean Biology29(1): 96–110. https://doi.org/10.1651/08-3020.1 CastresanaJ (2000) Selection of Conserved Blocks from Multiple Alignments for Their Use in Phylogenetic Analysis.Molecular Biology and Evolution17(4): 540–552. https://doi.org/10.1093/oxfordjournals.molbev.a026334 Campos-FilhoISDimitriouACTaitiSSfenthourakisS (2023) The genus Armadillo Latreille, 1802 (Oniscidea, Armadillidae) from Cyprus, with descriptions of two new species.Zootaxa5270(1): 67–91. https://doi.org/10.11646/zootaxa.5270.1.3 ChandlerCHBadawiMMoumenBGrèvePCordauxR (2015) Multiple Conserved Heteroplasmic Sites in tRNA Genes in the Mitochondrial Genomes of Terrestrial Isopods (Oniscidea). G3 Genes|Genomes|Genetics 5(7): 1317–1322. https://doi.org/10.1534/g3.115.018283 ChenGX (2000) Terrestrial Isopoda fauna of typical zones in China.Acta Zoologica Sinica46(3): 255–264. ChenGX (2003) Species Construction and Distribution of Terrestrial Isopoda in Typical Zones of China.Journal of Jishou University (Natural Science Edition)24(1): 14–19. DimitriouACTaitiSSchmalfussHSfenthourakisS (2018) A molecular phylogeny of Porcellionidae (Isopoda, Oniscidea) reveals inconsistencies with present taxonomy.ZooKeys801: 163–176. https://doi.org/10.3897/zookeys.801.23566 EberlR (2013) Phylogeography of the high intertidal isopod Ligia pallasii Brandt, 1833 (Isopoda: Oniscidea) from the Aleutian Islands to Monterey Bay.Journal of Crustacean Biology33(2): 253–264. https://doi.org/10.1163/1937240X-00002131 FolmerOBlackMHoehWLutzRVrijenhoekR (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.Molecular Marine Biology and Biotechnology3(5): 294–299. GentileGCampanaroACarosiMSbordoniVArganoR (2010) Phylogeography of Helleria brevicornis Ebner 1868 (Crustacea, Oniscidea): Old and recent differentiations of an ancient lineage.Molecular Phylogenetics and Evolution54(2): 640–646. https://doi.org/10.1016/j.ympev.2009.10.005 GentileGArganoRTaitiS (2022) Evaluating the correlation between area, environmental heterogeneity, and species richness using terrestrial isopods (Oniscidea) from the Pontine Islands (West Mediterranean).Organisms Diversity & Evolution22: 275–284. GongalskyKBNefedievPSTurbanovIS (2021) A new species of the genus Lucasioides Kwon, 1993 (Isopoda, Oniscidea, Agnaridae) from Siberia, Russia.Zootaxa4903(1): 140–150. https://doi.org/10.11646/zootaxa.4903.1.9 HeymannHNobleAC (1989) Comparison of canonical variate and principal component analyses of wine descriptive analysis data.Journal of Food Science54(5): 1355–1358. https://doi.org/10.1111/j.1365-2621.tb059 91.x. HijmansRJGuarinoLJarvisAO’BrienRMathurPBussinkCCruzMBarrantesIRojasE (2005) DIVA GIS. Available from: http://www.diva-gis.org (accessed 24 Jan. 2021) HurtadoLALeeEJMateosMTaitiS (2014) Global Diversification at the Harsh Sea-Land Interface: Mitochondrial Phylogeny of the Supralittoral Isopod Genus Tylos (Tylidae, Oniscidea). PLoS ONE 9(4): e94081. https://doi.org/10.1371/journal.pone.0094081 KalyaanamoorthySMinhBQWongTKFvon HaeselerAJermiinLS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates.Nature Methods14: 587–589. https://doi.org/10.1038/nmeth.4285 KarasawaS (2016) Eleven nominal species of Burmoniscus are junior synonyms of B. kathmandius (Schmalfuss, 1983) (Crustacea, Isopoda, Oniscidea).ZooKeys607: 1–24. https://doi.org/10.3897/zookeys.607.8253 KanazawaSKanazawaYKubotaK (2014) Redefinitions of Spherillo obscurus (Budde-Lund, 1885) and S. dorsalis (Iwamoto, 1943) (Crustacea: Oniscidea: Armadillidae), with DNA markers for identification. Edaphologia 93: 11−27. KashaniGM (2020) Not Porcellio nor Protracheoniscus but Lucasioides latus (Uljanin, 1875) (Isopoda, Oniscidea). Iranian Journal of Animal Biosystematics 16(1): 83−84. KatohKStandleyDM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability.Molecular Biology and Evolution30: 772–780. Khalaji-PirbaloutyVOraieHSantamariaCAWägeleJW (2022) Redescription of Tylos maindroni Giordani Soika, 1954 (Crustacea, Isopoda, Oniscidea) based on SEM and molecular data.ZooKeys1087: 123–139. https://doi.org/10.3897/zookeys.1087.76668 KlingenbergCP (2011) MorphoJ: An Integrated Software Package for Geometric Morphometrics.Molecular Ecology Resources11: 353–357. https://doi.org/10.1111/j.1755-0998.2010.02924.x Klossa-KiliaEKiliasGTryfonopoulosGKoukouKSfenthourakisSParmakelisA (2006) Molecular phylogeny of the Greek populations of the genus Ligidium (Isopoda, Oniscidea) using three mtDNA gene segments.Zoologica Scripta35(5): 459–472. https://doi.org/10.1111/j.1463-6409.2006.00243.x KumarSStecherGLiMKnyazCTamuraK (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35: 1547−154. KuznetsovaDMGongalskyKB (2012) Cartographic analysis of woodlice fauna of the former USSR.ZooKeys176: 1–11. https://doi.org/10.3897/zookeys.176.2372 KwonDH (1993) Terrestrial isopods from Korea. Korean Journal of Zoology 36: 133−158. KwonDH (1995) Terrestrial Isopoda (Crustacea) from Cheju Island, Korea. The Korean Journal of Systematic Zoology 11(4): 509−538. KwonDHTaitiS (1993) Terrestrial Isopoda from southern China, Macao and Hong Kong. Stuttgarter Beiträge zur Naturkunde, Series A 490: 1−83. LebedevYMGongalskyKBGorbunovAYZaitsevAS (2020) Rice Straw Decomposition by Woodlice (Isopoda, Oniscidea) and Millipedes (Myriapoda, Diplopoda) in the Soils of Kalmykia in a Laboratory Experiment.Arid Ecosystems10(3): 251–254. LeeTRC.HoSYWWilsonGDFLoN (2014) Phylogeography and diversity of the terrestrial isopod Spherillo grossus (Oniscidea: Armadillidae) on the Australian East Coast: Phylogeography of Spherillo grossus.Zoological Journal of the Linnean Society170(2): 297–309. https://doi.org/10.1111/zoj.12105 LetunicIBorkP (2021) Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research 49(W1): W293−W296. https://doi.org/10.1093/nar/gkab301 LiWC (2017) A new species of Lucasioides Kwon (Isopoda: Oniscidea: Agnaridae) from China. Zootaxa 4216(5): 495−500. https://doi.org/10.11646/zootaxa.4216.5.6 MarcadéICordauxRDoubletCDebenestVBouchonDRaimondR (2007) Structure and evolution of the atypical mitochondrial genome of Armadillidium vulgare (Isopoda, Crustacea).Journal of Molecular Evolution65: 651–659. https://doi.org/10.1007/s00239-007-9037-5 MinhBQSchmidtHAChernomorOSchrempfDWoodhamsMDvon HaeselerALanfearR (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era.Molecular Biology and Evolution37: 1530–1534. https://doi.org/10.1093/molbev/msaa015 MontesantoG (2015) A fast GNU method to draw accurate scientific illustrations for taxonomy. In: TaitiSHornungEŠtrusJBouchonD (Eds.) Trends in Terrestrial Isopod Biology.Zookeys515: 191–206. https://doi.org/10.3897/zookeys.515.9459 NunomuraN (1987) Studies on the terrestrial isopod crustaceans in Japan. IV. Taxonomy of the families Trachelipidae and Porcellionidae. Bulletin of the Toyama Science Museum 11: 1−76. NunomuraN (1991) Studies on the terrestrial isopod crustaceans in Japan. VI. Further supplements to the taxonomy. Bulletin of the Toyama Science Museum 14: 1−26. NunomuraN (2000) Terrestrial isopod and amphipod crustaceans from the Imperial Palace, Tokyo. Memoirs of the National Science Museum 35: 79−97. NunomuraN (2003a) Four new terrestrial isopod crustaceans from Kashima Islet and its neighboring, Tanabe Bay. Bulletin of the Toyama Science Museum 26: 13−24. NunomuraN (2003b) Terrestrial isopod crustaceans from southern Kyushu, southern Japan. Bulletin of the Toyama Science Museum 26: 25−45. NunomuraN (2008) Two new species of the genus Lucasioides (Crustacea, Isopoda) from mole’s tunnels of Nanto-shi, Toyama, middle Japan. Bulletin of the Toyama Science Museum 31: 3−11. NunomuraN (2010a) Terrestrial crustaceans from Shiga Prefecture, central Japan. Bulletin of the Toyama Science Museum 33: 47−63. NunomuraN (2010b) A new species of the genus Lucasioides (Crustacea: Isopoda) from Ashiu, Miyama, Nantan-shi, Kyoto, central Japan. Bulletin of the Toyama Science Museum 33: 39−45. NunomuraN (2010c) A new species of the genus Lucasioides (Crustacea: Isopoda) from nest material of Mogera imaizumii, Toyama-ken, central Japan. Bulletin of the Toyama Science Museum 33: 33−37. NunomuraN (2013a) Isopod crustaceans from Shikoku, western Japan-1, specimens from Echime Prefecture. Bulletin of the Toyama Science Museum 37: 19−78. NunomuraN (2013b) A new species of the family Agnaridae (Crustacea: Isopoda) from Kurehayama Hill, Toyama, middle Japan. Toyama Science Museum 445: 11−18. NunomuraNXieR (2000) Terrestrial isopod crustaceans of Yunnan, southwest China. In: Aoki J, Yin WY, Imadate G (Eds.) Taxonomical Studies on the Soil Fauna of Yunnan Province in Southwest China. Tokai University Press. Tokyo, 43−89. OliveiraJMMHenriquesIReadDSGweonHSMorgadoRG (2021) Gut and faecal bacterial community of the terrestrial isopod Porcellionides pruinosus: potential use for monitoring exposure scenarios.Ecotoxicology30: 2096–2108. ParmakelisAKlossa-KiliaEKiliasGTriantisKASfenthourakisS (2008) Increased molecular divergence of two endemic Trachelipus (Isopoda, Oniscidea) species from Greece reveals patterns not congruent with current taxonomy.Biological Journal of the Linnean Society95(2): 361–370. https://doi.org/10.1111/j.1095-8312.2008.01054.x PastorinoPBertoliMBrizioPCesarinaMANoraVDPrearoMPizzulE (2021) First insights into trace element accumulation by Philoscia affinis (Crustacea, Isopoda): a novel tracer to assess soil contamination in lowland plains? Biological Trace Element Research 199: 4782–4791. PoulakakisNSfenthourakisS (2008) Molecular phylogeny and phylogeography of the Greek populations of the genus Orthometopon (Isopoda, Oniscidea) based on mitochondrial DNA sequences.Zoological Journal of the Linnean Society152(4): 707–715. https://doi.org/10.1111/j.1096-3642.2007.00378.x PuillandreNLambertABrouilletSAchazG (2012) ABGD, Automatic Barcode Gap Discovery for primary species delimitation.Molecular Ecology21(8): 1864–1877. https://doi.org/10.1111/j.1365-294X.2011.05239.x. RanwezVHarispeSDelsucFDouzeryEJ (2011) MACSE: Multiple Alignment of Coding SEquences accounting for frameshifts and stop codons. Plos One 6(9): e22594. https://doi.org/10.1371/journal.pone.0022594 RatnasinghamSHebertPDN. (2007) BOLD: The Barcode of Life Data Systems.Molecular Ecology Notes7: 355–364. https://doi.org/10.1111/j.1471-8286.2007.01678.x RatnasinghamSHebertPDN (2013) A DNA-Based Registry for All Animal Species: The Barcode Index Number (BIN) System. PLoS ONE 8(7), e66213. https://doi.org/10.1371/journal.pone.0066213 RaupachMJRulikBSpeldaJ (2022) Surprisingly high genetic divergence of the mitochondrial DNA barcode fragment (COI) within Central European woodlice species (Crustacea, Isopoda, Oniscidea).ZooKeys1082: 103–125. https://doi.org/10.3897/zookeys.1082.69851 Rohlf FJ. tpsUTIL Version 1.56, TPSdig Version 2.17 (2013) Department of Ecology and Evolution, State University of New York at Stony Brook, New York. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539−542. https://doi.org/10.1093/sysbio/sys029 SchmalfussH (2003) World catalog of terrestrial isopods (Isopoda: Oniscidea).Stuttgarter Beiträge zur Naturkunde, Serie A,654: 1–341. SchmidtC (2002) Contribution to the phylogenetic system of the Crinocheta (Crustacea, Isopoda). Part 1 (Olibrinidae to Scyphaidae s. str.).Mitteilungen aus dem Museum für Naturkunde in Berlin (Zoologische Reihe)78: 275–352. SchmidtC (2003) Contribution to the phylogenetic system of the Crinocheta (Crustacea, Isopoda). Part 2 (Oniscoidea to Armadillidiidae).Mitteilungen aus dem Museum für Naturkunde in Berlin, Zoologische Reihe79: 3–179. SchmidtCLeistikowA (2004) Catalogue of genera of the terrestrial Isopoda (Crustacea: Isopoda: Oniscidea).Steenstrupia28: 1–118. SfenthourakisSTaitiS (2015) Patterns of taxonomic diversity among terrestrial isopods.ZooKeys515: 13–25. https://doi.org/10.3897/zookeys.515.9332 SongHBuhayJEWhitingMFCrandallKA (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proceedings of the National Academy of Sciences 105(36), 13486–13491. https://doi.org/10.1073/pnas.0803076105 TaitiSGruberGA (2008) Cave-dwelling terrestrial isopods from Southern China (Crustacea, Isopoda, Oniscidea), with descriptions of four new species. Research in South China karsts. Memorie del Museo Civico di Storia Naturale di Verona, Monografie Naturalistiche 3: 101−123. TsangLMMaKYAhyongSTChanTChuKH (2008) Phylogeny of Decapoda using two nuclear protein-coding genes: Origin and evolution of the Reptantia.Molecular Phylogenetics and Evolution48: 359–368. https://doi.org/10.1016/j.ympev.2008.04.009 UljaninA (1875) Rakoobrazniyia. Isopoda [Russian, with descriptions in Latin]. In: Fedtschenko A (Ed.) Puteschestvie v Turkestan, vol. 2. Zoogeografitcheskije Izsledovanija. St. Petersburg: 4−21, plates I−V. VandelA (1969) Results of the speleological survey in South Korea 1966. XIII. Isopodes terrestres récoltés dans les grottes de Corée. Bulletin of the national Science Museum 12: 157−159. WangJYangJBZengXGLiWC (2022) Integrative taxonomy on the rare sky-island Ligidium species from southwest China (Isopoda, Oniscidea, Ligiidae). BMC Zoology 7: 26. https://doi.org/10.1186/s40850-022-00120-1 WhitingMFCarpenterJCWheelerQDWheelerWC. (1997) The Strepsiptera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology.Systematic Biology46(1): 1–68. https://doi.org/10.1093/sysbio/46.1.1 WoRMS [World Register of Marine Species] (2024) Oniscidea. Available from: https://www.marinespecies.org/aphia.php?p=taxdetails&id=146505 (accessed 28 Feb. 2024) YangZ (2015) The BPP program for species tree estimation and species delimitation.Current Zoology61(5): 854–865. https://doi.org/10.1093/czoolo/61.5.854 YangXShaoMALiTC (2020) Effects of terrestrial isopods on soil nutrients during litter decomposition. Geoderma 376: 114546. YoshinoHKubotaK (2022) Phylogeographic analysis of Ligidium japonicum (Isopoda: Ligiidae) and its allied species reveals high biodiversity and genetic differentiation in the Kanto region, Japan. Entomological Science 25: e12501. https://doi.org/10.1111/ens.12501 ZengXGWangJYangJBLiWC (2021) Integrative taxonomy reveals a new species of the genus Burmoniscus (Isopoda, Philosciidae) from the Xuefeng Mountains, China.ZooKeys1055: 123–134. https://doi.org/10.3897/zookeys.1055.66879 ZhangJJKapliPPavlidisPStamatakisAA (2013) General Species Delimitation Method with Applications to Phylogenetic Placements.Bioinformatics29(22): 2869–2876. https://doi.org/10.1093/bioinformatics/btt499 ZhangFJantaritSNilsaiAStevensMIDingYSatasookC (2018) Species delimitation in the morphologically conserved Coecobrya (Collembola: Entomobryidae): A case study integrating morphology and molecular traits to advance current taxonomy.Zoologica Scripta47(3): 342–356. https://doi.org/10.1111/zsc.12279 ZhangDGaoFJakovlićIZouHZhangJLiWXWangGT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies.Molecular Ecology Resources20(1): 348–355. https://doi.org/10.1111/1755-0998.13096 Supplementary materials 10.3897/asp.82.e113041.suppl1 1E4A1367-2F1D-57F5-8117-D2024DEDBF20

Tables S1–S4

: .docx

Table S1. Description of the samples that were used for phylogenetic analysis, including taxon, DNA number, collection localities, DNA Data Bank of Japan (DDBJ) accession number, and Barcode Index Number (BIN) of the Barcode of Life Data System (BOLD). — Table S2. Percentage of divergence in the cytochrome c oxidase subunit I (COI) gene sequences of the Lucasioides species with outgroups. — Table S3. Percentage of divergence in the 18S rRNA gene sequences of the Lucasioides species with outgroups. — Table S4. Percentage of divergence in the 28S rRNA gene sequences of the Lucasioides species with outgroup.

 https://binary.pensoft.net/file/1083544 This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Wang J, Yao C-H, Jiang C, Li W-C (2024)