The Cryptic Diversity of the Terrestrial Microarthropods, Ptenothrix Börner (Collembola: Dicyrtomidae) from Taiwan: New Species Plus the Lectotype Designation for Ptenothrix denticulata (Folsom, 1899)

© 2024 Academia Sinica, Taiwan Open Access The Cryptic Diversity of the Terrestrial Microarthropods, Ptenothrix Börner (Collembola: Dicyrtomidae) from Taiwan: New Species Plus the Lectotype Designation for Ptenothrix denticulata (Folsom, 1899) Hsin-Ju Cheng1,§ , Bruno Cavalcante Bellini2,§ , Frans Janssens3, Taizo Nakamori4, and Chih-Han Chang1,5,* 1Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10617, Taiwan. *Correspondence: E-mail: [email protected], [email protected] (Chang) E-mail: [email protected] (Cheng) 2Department of Botany and Zoology, Biosciences Center, Federal University of Rio Grande do Norte (UFRN), Natal 59072-970, RN, Brazil. E-mail: [email protected] (Bellini) 3Evolutionary Ecology, Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium. E-mail: [email protected] (Janssens) 4Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan. E-mail: [email protected] (Nakamori) 5Department of Life Science, National Taiwan University, Taipei 10617, Taiwan lsid:zoobank.org:pub:16537856-6A02-48A7-928B-C8EB5353C0C4 §HJC and BCB contributed equally to this work. Received 3 May 2024 / Accepted 19 July 2024 / Published 12 November 2024 Communicated by John Wang This is the first taxonomic study of Collembola in Taiwan integrating morphological and molecular evidence to investigate the Taiwanese species in the genus Ptenothrix Börner. We discovered that specimens previously identified as Ptenothrix denticulata (Folsom, 1899) actually consist of several cryptic species, of which we described two species new to science. Our data revealed that, although these species are remarkably similar to each other, they can be distinguished by color patterns, chaetotaxic characters and DNA barcoding (COI). We also designated one of the syntypes of Ptenothrix denticulata (Folsom, 1899) as its lectotype, and treated Dicyrtoma denticulata (Salmon 1964) as a synonym of Ptenothrix denticulata (Salmon 1964) (syn. nov.). Lastly, our study suggests that the diversity of Collembola in Taiwan is still poorly understood, with a high potential for new studies focusing on these microarthropods. Key words: Soil fauna, Systematics, Genetic diversity, Phylogeny, Ptenothricinae BACKGROUND Collembola are one of the most abundant groups of terrestrial microarthropods that live in various habitats, including leaf litter, soil, plant surfaces, fungal sporocarps, decaying wood, etc (Bellinger et al. 1996– 2024). Within the litter-soil habitat, they are recognized as one of the most prevalent organisms, often attaining densities of up to 40,000 individuals per square meter in temperate grasslands or forests (Orgiazzi et al. 2016). To date, approximately 9,500 extant Collembola species have been described globally, with estimates Citation: Cheng HJ, Bellini BC, Janssens F, Nakamoriv T, Chang CH. 2024. The cryptic diversity of the terrestrial microarthropods, Ptenothrix Börner (Collembola: Dicyrtomidae) from Taiwan: new species plus the lectotype designation for Ptenothrix denticulata (Folsom, 1899). Zool Stud 63:42. doi:10.6620/ZS.2024.63-42. Zoological Studies 63:42 (2024) doi:10.6620/ZS.2024.63-42 1 © 2024 Academia Sinica, Taiwan suggesting that the group species richness may actually reach 50,000 or even more (Hopkin 1997; Bellinger et al. 1996–2024). In spite of past endeavors, a large amount of the Collembola diversity is still unknown, particularly in countries that lack a history of taxonomic research of these taxa, such as Taiwan. Dicyrtomidae Börner, 1906 (sensu Deharveng 2004) is a family of springtails from the order Symphypleona, and its species are mostly atmobiotic (Bretfeld 1999), inhabiting plant leaf and litter surfaces. Their body is about 1 to 3 millimeters long and pigmented (some species show very distinct color patterns), and they have long limbs and a full set of eight ocelli per side of the head. The main features that separate the Dicyrtomidae from other Symphypleona families are aspects of the antennae, which have an antennomere IV shorter than half of III, and antennae elbowed between antennomeres II and III (Bretfeld 1999). Currently, Dicyrtomidae comprises two subfamilies: Ptenothricinae Richards, 1968, which features the presence of bothriotrichum D in adults, and Dicyrtominae Richards, 1968, which lacks this characteristic (Bretfeld 1999). Although Dicyrtomidae are colorful and have relatively large body sizes, not many in-depth studies have applied integrative and modern methods to explore the diversity of this family. The lack of fundamental studies has significantly hindered our understanding on the biology and ecology of dicyrtomids, for instance, the underlying mechanisms of their coloration polymorphism. In Asia, studies related to Dicyrtomidae have not seen much progress after the 1970s (e.g., Yosii 1970; Yosii and Lee 1963). These past studies were mainly based on morphology and focused on taxonomy. After this, little molecular and other complementary evidence has been used to study this fauna (e.g., Cheng et al. 2022). This lack of effort has greatly underestimated the Dicyrtomidae species diversity, especially when cryptic species exist. In addition, morphological polymorphism, e.g., color pattern variations, could be misleading since species identification is only based on morphological characteristics, and previous studies of dicyrtomids lack other useful diagnostic details applied in the current taxonomy of Symphypleona (Mateos 2008; Katz et al. 2015; Potapov et al. 2018). In Taiwan, only seven species of Dicyrtomidae have been recorded so far (Cheng et al. 2022). However, the diversity of Taiwanese Dicyrtomidae clearly surpasses the existing records (H.-J. Cheng, pers. obs.), and more research is needed to better circumscribe this family. Ptenothrix is the most species-rich genus in Dicyrtomidae, with about 80 described extant species (Bellinger et al. 1996–2024). Compared to other Dicyrtomidae, the main diagnostic characteristics of the genus include: (1) head dorsally with prominent spinelike chaetae; (2) large abdomen without protuberance, but with the full set of bothriotricha (A–D), and posteriorly dorsally covered by rows of small spinelike chaetae; (3) neosminthuroid chaetae absent from the parafurcal area; (4) posterior side of hind tibiotarsus with 2 (rarely 3) modified serrated chaetae; (5) claws usually without a tunica; (6) tenaculum with 4+4 teeth; and (7) outer and inner chaetae of dens often coarsely serrated (Yosii and Lee 1963; Yosii 1970; Bretfeld 1999; Fjellberg 2007; Oliveira et al. 2020; Jo and Park 2023). So far, only three species of Ptenothrix have been documented in Taiwan: Ptenothrix denticulata (Folsom, 1899), Ptenothrix corynophora Börner, 1909, and Ptenothrix monochroma Yosii & Lee, 1963 (Cheng et al. 2022). The number of described Ptenothrix spp. in Taiwan is fewer than those of neighboring countries; for example, at least 17 exist in Japan (Ito et al. 2012) and seven in Korea (Jo and Park 2023). Apparently, the diversity of Ptenothrix in Taiwan is poorly understood. Furthermore, many Asiatic species of the genus are remarkably similar to each other in overall chaetotaxy and even in color pattern (Folsom 1899; Börner 1909; Yosii and Lee 1963; Lin and Xia 1985a b), a condition that raises doubts about the validity of some of these species, or contrarily, supports the presence of cryptic diversity within the genus. This study aims to investigate the cryptic diversity of Ptenothrix in Taiwan. We hypothesize that specific color patterns can be reliable diagnostic characteristics of morphologically similar species in the genus. The differences of color pattern on the head vertex of specimens in Taiwan made us hypothesize that they are not the recorded Ptenothrix denticulata (Folsom, 1899), but are actually two new and distinct species. MATERIALS AND METHODS Specimen collection Specimens of Ptenothrix were collected throughout Taiwan during 2021–2023 (Fig. 1). The collection methods included beating vegetation and aspirating from grass blades of Miscanthus, leaves of Bambusoideae, dead leaves of understory vegetation, and rotten wood (Fig. 2), as well as setting Malaise traps. Specimens were preserved in 85% ethanol and stored at 4°C until morphological examination and molecular analysis. In order to confirm the identities of the sampled specimens, two type specimens of Ptenothrix denticulata (Folsom, 1899) deposited at the Smithsonian National Museum of Natural History page 2 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan Fig. 1. Sampling localities of the two new species in Taiwan. Red dots represent Ptenothrix taiwanensis sp. nov. sampling sites, mainly distributed in north and central Taiwan and one locality in Nangan Island in Matsu, which could be due to recent human intervention. Green triangles represent Ptenothrix matsuensis sp. nov., with known distribution only in Nangan Island, Matsu. Fig. 2. In situ images of specimens of Ptenothrix taiwanensis sp. nov., indicating that rotten wood and algae-covered surface are two of their preferred habitats. page 3 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan (NMNH), Washington, USA (USNMENT01747048) and the Museum of Comparative Zoology in Harvard University (MCZ 14770) were examined (Fig. 3). Additionally, we attempted to collect topotype specimens of Ptenothrix denticulata (Folsom, 1899) in its type locality in Japan (Komaba, Tokyo). Specimens that were newly collected and analyzed in this study were deposited at the NTU Insect Museum, Department of Entomology, National Taiwan University (NTUE), and the National Museum of Natural Science in Taichung, Taiwan (NMNS). Molecular analysis For molecular phylogenetic analysis, DNA was extracted from entire specimens using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The specimens were subjected to an overnight incubation at 56°C subsequent to the addition of ATL buffer containing proteinase K. After this, 1 μl of carrier RNA was introduced into buffer AL. The resultant DNA-containing buffer was transferred into a fresh column. Simultaneously, the voucher specimens were preserved in a solution of 75% ethanol after DNA extraction so as to link morphology with genetic data. After extraction, the DNA was eluted in a 50 μl buffer and subsequently stored at -20°C. The mitochondrial marker cytochrome c oxidase subunit 1 (COI) was chosen for phylogenetic analysis. Polymerase chain reactions (PCRs) were conducted using primers Fig. 3. Syntypes of Ptenothrix denticulata. (A) Slide specimen USNMENT01747048 from the Smithsonian National Museum of Natural History; arrow indicates that Salmon intended to designate it as the lectotype. (B) Ethanol specimen MCZ 14770 from the Museum of Comparative Zoology at Harvard University (the picture was provided by the museum); the arrow indicates the presence of a transversal band of pigment on the head vertex. page 4 of 25 Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan LCO1490 and HCO2198 (Folmer et al. 1994), and the PCR cycles followed Cheng et al. (2022). The obtained sequences were assembled using Geneious (Biomatters Ltd., Auckland, New Zealand), double-checked by eye, and aligned employing ClustalX 2.0 (Larkin et al. 2007). Recorded species of Dicyrtomidae in Taiwan and neighboring regions, including Ptenothrix corynophora Börner, 1909, Ptenothrix monochroma Yosii & Lee, 1963, Ptenothrix huangshanensis Chen & Christiansen, 1996, Papirioides jacobsoni Folsom, 1924, and Papirioides caishijiensis Wu & Chen, 1996, were included in the phylogenetic analyses because they are the only species that have reliable COI sequences on GenBank. Sminthurus viridis (Linnaeus, 1758) (Sminthuridae, Symphypleona) was used as the outgroup. Kimura’s two-parameter distances of the aligned sequences were calculated with MEGA X (Kumar et al. 2018). Maximum Likelihood analysis was performed employing IQ-TREE (Nguyen et al. 2015). The selection of optimal partitioning schemes and molecular evolution models for the phylogenetic analyses was conducted using PartitionFinder (Lanfear et al. 2016). Clades exhibiting p-distances of COI exceeding 8% were tentatively designated as potential species following the approach outlined by Katz et al. (2015). These findings were subsequently integrated with morphological characters to facilitate species delimitation. Examining slide specimens The ethanol-preserved specimens were clarified with Nesbitt’s fluid (chloral hydrate 40 g, distilled water 25 ml, concentrated hydrochloric acid 2.5 ml), cleaned with Arlé’s fluid (chloral hydrate 9 g, 85% lactic acid 9 ml, glycerin 9 g, 9 drops of formalin) and mounted in Hoyer’s medium (gum arabic 6 g, chloral hydrate 40 g, glycerin 4 ml, distilled water 10 ml) (Arlé and Mendonça 1982; Bellinger et al. 1996–2024). Slide and ethanol-preserved specimens were examined using a Nikon SMZ800N stereomicroscope equipped with a plan Apo 1× objective lens and a Nikon ECLIPSE E200 compound microscope and photographed using a TOUPCAM MTR3CMOS07100KPA digital camera. For the chaetal terminology, we followed: Betsch and Waller (1994) for the head and large abdomen chaetotaxy; Fjellberg (1999) for the labial palp; Cipola et al. (2014) for the labral chaetotaxy; Betsch (1997) for the small abdomen, also using the labels provided by Yosii and Lee (1963), as an alternative description for comparison between the new species and other described ones; and Bretfeld (2001) for dorsal chaetae of the dens. Chaetae labels are marked in bold in the text. Abbreviations used in descriptions are: Ant— antennal segment(s), Abd—abdominal segment(s), Th— thoracic segment(s). Body and appendages covered by different types of chaetae are represented in figure 4. Examining specimens with SEM For scanning electron microscopy (SEM), specimens were dehydrated (Schneeberg et al. 2017) and dried at critical point using a Hitachi HCP-2 Critical Point Dryer. Dried specimens were glued on a carbon conductive tape, sputter coated with platinum, and mounted on a specimen holder. Finally, images were captured using a Hitachi Tabletop TM-3000 scanning electron microscope under Hitachi TM3000 software. RESULTS Examination of the archived types of Ptenothrix denticulata suggests that in 1899, Folsom described the species based on three type specimens without designating a holotype (Folsom 1899). Therefore, in accordance with ICZN Article 73.2, all three specimens of the type series can be automatically considered syntypes. According to Salmon (1964: 634 as Dicyrtoma and p. 642 as Ptenothrix), the specimens of the type series have been preserved: one as holotype and as Dicyrtoma in the Museum of Comparative Zoology in Harvard University, USA (now missing), and two as paratypes, one in the Smithsonian Fig. 4. Chaetae symbols used in the morphological descriptions of the new species. page 5 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan National Museum of Natural History, Washington, USA (USNMENT01747048; Fig. 3A) and another in the Museum of Comparative Zoology in Harvard University, USA (MCZ14770; Fig. 3B). The slide specimen USNMENT01747048 is not in ideal condition and the gut contents greatly decrease the visibility of its chaetotaxy, making it impossible to verify its morphology in detail. Using the images of the ethanol specimen MCZ14770 provided by the Museum of Comparative Zoology at Harvard University, we were able to confirm that there is a continuous transverse stripe behind the eyes, which is consistent with the figures in the original description (Folsom 1899). However, this specimen is severely deformed, and it is impossible to examine further characteristics in detail. Therefore, although the two specimens of the type series exist, it is not possible to study their morphology in detail to redescribe the species. A redescription based on more recent materials from the type locality is urgently needed. Fourteen Ptenothrix specimens from the type locality of Ptenothrix denticulata (Folsom, 1899) in Japan (Komaba, Tokyo) were examined, and all specimens have a discontinuous transverse stripe on the vertex of the head, consistent with the original description of P. denticulata var. catenata Börner, 1909. However, according to the original description and the type specimen deposited at Harvard University, Ptenothrix denticulata has a continuous transverse stripe on the head vertex. Thus, the specimens collected from the type locality were identified as P. denticulata var. catenata. According to Article 45.6.4 of ICZN, since Ptenothrix denticulata var. catenata was published before 1961, and the author explicitly used the term “var.”, it is now a subspecies: Ptenothrix denticulata catenata Börner, 1909. Among the 67 specimens collected throughout Taiwan, we recognized two morphospecies that are morphologically similar to Ptenothrix denticulata (Fig. 5A–F). However, we were unable to find any P. denticulata topotypes in the type locality in Japan after several attempts. Based on the original description and re-description of P. denticulata (Folsom, 1899; Yosii and Lee 1963), we discovered that the two morphospecies differ considerably from P. denticulata in the dorsal head patterns and the number of teeth on the basal unguiculus. The mean interspecific K2P distance between the two morphospecies was 16.7%, comparable to the interspecific genetic variations of the analyzed Ptenothrix specimens (14.0–17.5%; Table 1). Additionally, both morphospecies were recovered as independent monophyletic lineages in the maximum likelihood analysis (Fig. 6). Thus, based on both morphological and molecular results, we concluded that the two morphospecies are indeed two species new to science, namely Ptenothrix taiwanensis sp. nov. and Ptenothrix matsuensis sp. nov. Ptenothrix taiwanensis sp. nov. was mainly found in the low elevations in north and central Taiwan, whereas P. matsuensis sp. nov. was collected from Nangan Island in Matsu during the wet season (Table S1). The two new species are described below. Order Symphypleona Börner, 1901 sensu Bretfeld, 1986 Suborder Appendiciphora Bretfeld, 1986 Superfamily Dicyrtomoidea Börner, 1906 sensu Bretfeld, 1994 Family Dicyrtomidae Börner, 1906 Subfamily Ptenothricinae Richards, 1968 sensu Bretfeld, 1999 Genus Ptenothrix Börner, 1906 Genus diagnosis: Large abdomen without protuberance. Head, large abdomen anteriorly, and Abd VI with large spines (blunt acanthoid chaetae). Large abdomen posteriorly with longitudinal rows of short spines. Bothriotrichia A and D present on adults. Neosminthuroid chaetae missing on the parafurcal area. Dorsal valve of small abdomen dorsally with 5 chaetae arranged in two transverse rows; anterior with 3 chaetae and posterior with 2 robust, large and apically rounded chaetae (blunt acanthoid chaetae). Tibiotarsus III posteriorly with 2 (or rarely 3) modified serrated chaetae. Claws mostly without a tunica. Retinaculum with 4+4 teeth. Outer and inner dorsal chaetae of dens often serrated (based on Yosii and Lee 1963; Yosii 1970; Bretfeld 1999; Fjellberg 2007; Oliveira et al. 2020; Jo and Park 2023). Type species: Podura atra Linnaeus, 1758. Ptenothrix taiwanensis sp. nov. (Figs. 5A–C, 7–11, 12A, 12C, 13, Table 2) urn:lsid:zoobank.org:act:DC999C0A-A464-458B-99F00E65B70FD2CB Ptenothrix denticulata (Folsom, 1899): Cheng et al. 2022, p. 137–138 (text-fig. 4A). Type material: TAIWAN: Holotype ♀ on slide: Taiwan, New Taipei City, Xindian Dist., Changfu Temple (24.91491, 121.53243), 7, May. 2022. by searching rotten wood, HJ Cheng, DY Cheng, JK Cheng leg. (NTUE13100001). 4 paratypes on slides: 3♀1♂, same data as holotype (1♀1♂ in NTUE: ♀ NTUE13100002, ♂ NTUE13100003; 2♀in NMNS: 8848-1, 8848-2). 3 paratypes in ethanol: 2♀1 sex undetermined (DNA voucher), same data as holotype page 6 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan (1♀1 sex undetermined in NTUE: ♀ NTUE13100004, sex undetermined NTUE13100005; 1♀ in NMNS: 8848-3). Other analyzed slide specimens: TAIWAN: 1♀, Keelung City, Anle Dist., Lover’s Lake Park (25.159123, 121.704894), 23. September. 2022, by searching rotten wood, leg. HJ Cheng, ZJ Chang (NTUE13300001); 1♀, Yilan County, Datong Township (24.619015, 121.679917), 1. October. 2022, by beating vegetation, leg. HJ Cheng, ML Fu (NMNS: 8848-4); 2♂, Pingtung County, Manzhou Township, 31. June to 4. July. 2009, leg. ML Jeng (NMNS: 8848-5, 8848-6); 3♀1♂, Hualien County, Da Nong Da Fu Forest Park, 18. November to 12. December. 2018, flight interception Fig. 5. Habitus of the two new species and subspecies catenata of the P. denticulata group : (A, B, C) Dorsal, lateral, and facial views of Ptenothirx taiwanensis sp. nov., respectively; (D, E, F) Dorsal, lateral, and facial views of Ptenothrix matsuensis sp. nov., respectively; (G, H, I) Dorsal, lateral, and facial of Ptenothrix denticulata catenata Börner, 1909, respectively. page 7 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan Fig. 6. Maximum Likelihood tree based on COI sequences of Dicyrtomidae species in Taiwan. Genera Papirioides Folsom, 1924 and Ptenothirx are labeled with blue and yellow backgrounds, respectively. The red bar indicates the clade of Ptenothrix taiwanensis sp. nov.; the green bar indicates the clade of Ptenothrix matsuensis sp. nov.. Node values represent the ultrafast bootstrap support (≥ 90%) and SH-aLRT test (≥ 80%) values, respectively. Sminthurus viridis (Sminthuridae, Symphypleona) represents the outgroup. page 8 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan trap, leg. YH Ho (NMNS: ♀8848-7, ♀8848-8, ♀8848-9, ♂8848-10); 1♀3♂, Tainan City, Dongshan Dist., Shihe Mountain, 27. August to 7. October. 2021, Malaise trap, leg. ML Jeng, YH Ho, WT Yang (NMNS: ♀8848-11, ♂8848-12, ♂8848-13, ♂884814); 2♀1♂ New Taipei City, Fushan Botanical Garden (24.7634, 121.5849), 14. September to 2. October. 2021, flight interception trap, leg. YH Ho, CT Hsu (NMNS: ♀8848-15, ♀8848-16, ♂8848-17); 1♀, Hsinchu County, Syakaro National Trail, 18. May. 2022, leg. LH Chen (NTUE13300002). Etymology: The name taiwanensis, an adjective meaning “Taiwanese”, is given to this species with reference to its type locality, Taiwan. Diagnosis: Head vertex with a median longitudinal stripe of dark pigment between the eyes, each eyepatch with a posterior dark spot (Fig. 5A, C), dorsal large abdomen with a posterior dark spot, small abdomen without any large spot of pigment (Fig. 5A). Ant II with 3 (Fig. 7B), Ant III with 10 cup sensilla, distal Ant III partially divided into about 8 pseudo-subsegments (Fig. 7C), Ant IV with 4 subsegments (Fig. 7D). Head frontal area with 6+6 spines, D line central spines shorter than the lateral ones, clypeal area with 2 large blunt unpaired central acanthoid chaetae and 2 less thick midfacial unpaired chaetae, b, c, e and f lines with 2 cup sensilla each (Fig. 8A). Large abdomen anterior Th. II m line with 1+1 or 2+2 large blunt acanthoid chaetae, dorsoposterior region with 4 longitudinal lines of short spines, with 6, 5, 14, 14 spines from the more lateral to the more dorsal line, respectively (Fig. 9A). Parafurcal area with two cup sensilla, without any serrated chaetae. Dorsal anal valve of small abdomen with as1–2, ms2, ms4 and mps4 as blunt acanthoid chaetae in both sexes; ventral anal valve aai1 of the female and mi1 of both sexes as blunt acanthoid chaetae, 3 chaetae between aai1 and ai6 in males and females; female subanal appendage smooth, blunt and thick (Fig. 9B, C). Tibiotarsus III with 2 modified serrated chaetae, 1 microsensillum in a cavity (oval organ), 5 cup sensilla, and 4 oval organs (without inner sensilla) (Fig. 10F). Ungues with pseudonychia and lacking tunica, with 2 dorsal, 2 internal and 2+2 lateral teeth each, unguiculi with one internal tooth each, apical filaments acuminate or slightly knobbed, surpassing the ungues tip on all legs (Fig. 11A–C). Manubrium with 8–10 dorsal chaetae on each side. Dens dorsally (posteriorly) with 24 chaetae, 15 of them feathered (Fig. 11F), dens ventral (anterior) formula from the apex to the basis: 3, 2, 1, 1… 1 (Fig. 11G). Description: Average body (head + trunk) length of type series: 2.4 mm in females and 1.77 mm in males, holotype with 2.3 mm. Habitus typical of the genus. Body and appendages with different types of chaetae as presented in figure 4. Specimens in ethanol with background color white to pale yellow, with mottled brown symmetrical longitudinal stripes upon the anterior 4/5 of the dorsum, connected by a stripe and scattered around lateral sides of the body. A dark almost square patch on the bulge of the dorso-posterior large abdomen, small abdomen without any large spot of pigment, head with irregular patterns of pigment, vertex with a longitudinal stripe of dark pigment between eyes, each eyepatch with a posterior dark spot. Ant I redpurple, Ant II basally pale red, apically dark purple, Ant III–IV dark purple. Legs yellowish, banded with three purple stripes on tibiotarsus. Furca pale (Fig. 5A, B). Most body chaetae smooth (not denticulate). Head (Figs. 7–8): Antennae shorter than body, 1.7 mm in holotype. Holotype antennal segment ratio of Ant I:II:III:IV as 1:6.26:8.28:1.57. Ant I with 7 regular chaetae, two of them ventral (Fig. 7A), Ant II with 20 regular chaetae plus 3 cup sensilla (Fig. 7B), Ant III distally partially divided into about 8 distal pseudo-subsegments, with 80 regular chaetae, 10 cup sensilla and 1 microsensillum, apical organ typical, with 2 sense rods inside a single shallow invagination (Fig. 7C), Ant IV with 4 subsegments and about 61 Table 1. Mean interspecific and intraspecific (grey background) K2P distances of COI sequences of the Dicyrtomidae and Sminthuridae species in the phylogenetic analysis 123456789 1. Ptenothrix taiwanensis sp. nov. 5% (n = 26) 2. Ptenothrix matsuensis sp. nov. 16.7% 0% (n = 5) 3. Ptenothrix denticulata catenata 16.7% 16.0% 1% (n = 6) 4. Ptenothrix corynophora 13.3% 14.0% 16.3% 5% (n = 15) 5. Ptenothrix monochroma 17.5% 14.1% 16.9% 14.8% 7% (n = 4) 6. Ptenothrix huangshanensis 16.3% 14.2% 16.5% 14.7% 15.0% 0% (n = 1) 7. Papirioides jacobsoni 20.9% 19.9% 19.9% 20.6% 18.9% 20.3% 7% (n = 14) 8. Papirioides caishijiensis 19.2% 19.0% 18.4% 17.2% 17.3% 20.0% 18.7% 0% (n = 1) 9. Sminthurus viridis 26.4% 28.5% 24.5% 28.7% 29.0% 28.2% 27.6% 28.3% 0% (n = 2) page 9 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan (Fig. 11E); dens dorsally (posteriorly) with 24 chaetae: 10 on the dorso-external (E1–10), 10 on the dorsointernal (J1–10) and 4 dorso-central (PE1–4) chaetae, respectively, external and internal chaetae feathered (Fig. 13C), except for the chaetae near the manubrium and an apical-external one (Fig. 11F); dens ventrally (anteriorly) with 8 chaetae, with the following formula from the apex to the basis: 3, 2, 1, 1… 1 (Fig. 11G); mucro with a narrow apex, with both edges serrated, with about 33 (inner) and 28 (outer) serrations on each edge, with an apical notch (Fig. 11H). Ratio mucro: dens: manubrium in holotype 1: 3.3: 1.05. Distribution: Low elevation of Taiwan (mainly the northern and central region), and one locality in Nangan Island, Matsu (public toilet of Mt. Yuntai). Habitat: Most Dicyrtomidae, including Ptenothrix taiwanensis sp. nov. are atmobiotic (Bretfeld 1999). Specimens of the new species generally inhabit wet rotten wood and dead leaves of tree ferns and Miscanthus bushes. Some specimens were also found associated with the leaf litter layer, especially juveniles. Sampling localities of Ptenothrix taiwanensis sp. nov. are presented as orange dots in figure 1. Remarks: Ptenothrix taiwanensis sp. nov. specimens were identified as Ptenothrix denticulata (Folsom, 1899) in Cheng et al. (2022), which is now proven to be a misidentification (see Fig. 6 and Table 2). Ptenothrix taiwanensis sp. nov. is remarkably similar Fig. 12. Morphological comparison of the two new species. Frontal spines on line D of: (A) Ptenothrix taiwanensis sp. nov.; and (B) Ptenothrix matsuensis sp. nov. (white arrows mark the central spines, black arrows mark the lateral spines near the eyes). Small abdomen of a male of: (C) Ptenothrix taiwanensis sp. nov.; and (D) Ptenothrix matsuensis sp. nov. The latter species lacks one chaeta between aai1 and ai6 (marked with a dotted circle in panel D) compared to the former one. page 16 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan Fig. 13. Ptenothrix taiwanensis sp. nov. SEM (scanning electronic microscope) images: (A) Bothriotrichum A of the large abdomen; (B) Cup sensillum on tibiotarsus; (C) Serrated dental chaetae; (D) Oval organ on tibiotarsus; (E) Microsensillum on tibiotarsus; (F) Modified serrated chaeta on tibiotarsus III; (G) Right eyepatch, dorsal view; (H) Collophore vesicle with warty sacs; (I) Collophore sacs with warty vesicles at ventral view; (J) Tenaculum, lateral view; (K) Lateral view of the empodial complex I, unguis with 2 unpaired inner (black arrows) and 2 lateral teeth; (L) Dorsal view of unguis I, showing the pseudonychia merged with the dorsal unguis, at the apex there is the unguiculus elongated axial filament, slightly knobbed at the tip. page 17 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan to Ptenothrix matsuensis sp. nov., described in the next section. We consider both to be cryptic species due to their strong resemblance in color pattern and overall chaetotaxy. In addition to Taiwan Island, P. taiwanensis was also collected on the tile wall in a public toilet in Nangan Island in Matsu, but was not found in any other localities in Matsu. This made us presume that the record in Matsu could be due to recent human intervention. Compared with other Ptenothrix species found in the vicinities specimen collection, such as Taiwan and South China, Japan, and the type locality of P. denticulata (Table 2), the two new species differ from the others by: frontal head A internal spines as long as lateral ones (shorter than the lateral ones in P. denticulata (Folsom, 1899) and longer in P. denticulata catenata Börner, 1909, P. corynophora Börner, 1909 P. gigantisetae Lin & Xia, 1985, and P. huangshanensis Chen & Christiansen, 1996), Ant IV with 4 subdivisions (2–4 in P. denticulata, 2–3 in P. corynophora, 5 in P. huangshanensis, and 7 in P. gaoligonshanensis Itoh & Zhao, 2000), and dorsal dens with 10 inner chaetae (9 in P. annulatus, P. dinghuensis, P. gigantisetae, P. palmisetacea, and P. sinensis). Further comparisons are listed in table 2. Table 2. Main differences between the Ptenothrix species found in Taiwan, Japan, and South China Species Head vertex color pattern Ant IV subdivisions Ant III subdivisions Frontal head A internal spines compared to the lateral ones Frontal head D internal spines compared to the lateral ones Total n° of large abdomen dorsal spines P. taiwanensis sp. nov. A patch behind each eye, a central longitudinal stripe 4 ± 8 = < 39 P. matsuensis sp. nov. A patch behind each eye, an indistinct central patch (sometimes absent) 4 ± 8 = = 34–39 P. denticulata A transverse band 41; 2–32At least 91; 5–62<2<211? P. denticulata catenata A patch behind each eye344± 84>4<4? P. corynophora No patch625, 3665; 76> =6? P. monochroma No patch 4 ? ? ? ? P. annulatus Numerous yellow round patches unclear 5 ? ? ? P. dinghuensis ? 4 6 ? < ? P. gigantisetae No patch 4 7 > > ? P. huangshanensis Head with a quadrilateral hollow spot posterior to each eyepatch 5 8 > = 34 P. palmisetacea ? 4 7 ? ? ? P. yunnanus A broad median streak on the vertex 4 7 > < ? P. gaoligonshanensis ? 7 7 = < ? P. sinensis ? 4 7 ? ? ? Species Unguiculus III teeth Manubrial dorsal chaetae Inner chaetae row of dorsal dens Outer chaetae row of dorsal dens N° of outer teeth of mucro N° of inner teeth of mucro References P. taiwanensis sp. nov. 1 8–10 10 10 28 33 This study P. matsuensis sp. nov. 1 7 10 10 34 46 This study P. denticulata 21, 12? 102102312382 1Folsom 1899; 2Yosii and Lee 1963 P. denticulata catenata 1 74104104? ? 3Börner 1909; 4This study P. corynophora 16> 7 (10?)6? ? 26 42 5Börner 1909; 6Yosii and Lee 1963 P. monochroma 1 9 ? ? 47 36 Yosii and Lee 1963 P. annulatus 1 ? 9 9 36 45 Lin and Xia 1985a P. dinghuensis 0 ? 9 10 22 26 Lin and Xia 1985a P. gigantisetae 1 ? 9 9 21 24 Lin and Xia 1985b P. huangshanensis 1 8 10 10 21–29 28–31 Chen and Christiansen 1996 P. palmisetacea 1 ? 9 10 18–20 18–20 Lin and Xia 1985a P. yunnanus 1 8 ? ? 26 22 Itoh and Zhao 2000 P. gaoligonshanensis 1 8 10 10 42 30 Itoh and Zhao 2000 P. sinensis 1 ? 9 10 32 26 Lin and Xia 1985a page 18 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan Ptenothrix matsuensis sp. nov. (Figs. 5D–F, 12B,12D, 14, Table 2) urn:lsid:zoobank.org:act:B784F1E5-234C-4B42-8F29F6B96A9AE17A Type materials: TAIWAN: Holotype ♀ on slide, Lienchiang County, Nangan Township, Shengtian Park (26.154204, 119.916068), 25. May. 2023, by beating vegetation, leg. HJ Cheng, YH Ho (NTUE13100006). 4 paratypes on slides: 2♀2♂, same data as holotype (1♂1♀ in NTUE: ♂ NTUE13100008, ♀ NTUE13100007, 1♂1♀ in NMNS: ♂ 884818, ♀8848-19). 2 paratypes in ethanol: 1♂1 sex undetermined (DNA voucher), Lienchiang County, Nangan Township, Motianling (Skyscraper Ridge) Trail (26.161192, 119.953177), 25. May. 2023, by beating vegetation, leg. HJ Cheng, YH Ho (1♂1 sex undetermined in NTUE: ♂ NTUE13100009, sex undetermined NTUE13100010). Other analyzed slide specimens: TAIWAN: 3♀ on slide, Lienchiang County, Nangan Township, Siwei Rd. (26.173417, 119.917225), 25. May. 2023, leg. HJ Cheng, YH Ho (2 in NTUE: NTUE13300003 and NTUE13300004; 1 in NMNS: 8848-20). 3♂ on slide, Lienchiang County, Nangan Township, Motianling (Skyscraper Ridge Trail) (26.161192, 119.953177), 25. May. 2023, leg. HJ Cheng, YH Ho (1 in NTUE: NTUE13300005; 2 in NMNS: 8848-21, 8848-22). Etymology: The new species was named after its type locality, Matsu Islands. Diagnosis: Head vertex without a longitudinal stripe of dark pigment between eyes, each eyepatch with a posterior dark spot (Fig. 5D), dorsal large abdomen lacking any dark spot, dorsal small abdomen almost entirely covered by a large patch of dark pigment (Fig 5D). Ant II with 3, Ant III with 10 cup sensilla, distal Ant III partially divided into about 8 pseudosubsegments, Ant IV with 4 subsegments. Head frontal area with 6+6 spines, D line central spines as long as the lateral ones (Figs. 12B, 14A), clypeal area with 2 large blunt unpaired central acanthoid chaetae and 2 less thick midfacial unpaired chaetae, b, c, e and f lines with 2 cup sensilla each. Large abdomen Th. II anterior m line with 1+1 large blunt acanthoid chaetae, dorso-posterior region with 4 longitudinal lines of short spines, with 5–6/ 5/ 12–14/ 12–14 spines from the more lateral to the more dorsal line, respectively (Fig. 14B). Parafurcal area with two cup sensilla, without any serrated chaetae. Dorsal anal valve of small abdomen with as1–2, ms2, ms4 and mps4 as blunt acanthoid chaetae in both sexes; ventral anal valve aai1 of the female and mi1 of both sexes as a blunt acanthoid chaetae, 3 chaetae between aai1 and ai6 in females, only 2 in males (Figs. 12D, 14C); female subanal appendage smooth, blunt and thick. Tibiotarsus III with 2 modified serrated chaetae, 1 microsensillum in a cavity (oval organ), 5 cup sensilla, and 4 oval organs (without inner sensilla) (as in Fig. 10F). Ungues with pseudonychia and lacking tunica, with 2 dorsal, 2 internal and 2+2 lateral teeth each, unguiculi with one internal tooth each, apical filaments acuminate or slightly knobbed, surpassing the ungues tip in all legs (as in Fig. 10A–C). Manubrium with 7 dorsal chaetae on each side (Fig. 14D). Dens dorsally (posteriorly) with 24 chaetae, 15 of them feathered (as in Fig. 11F), dens ventral (anterior) formula from the apex to the basis: 3, 2, 1, 1… 1 (as in Fig. 11G). Description: Average body (head + trunk) length of type series: 1.82 mm in females and 1.38 mm in males, holotype with 1.9 mm. Habitus typical of the genus. Overall chaetotaxy similar to Ptenothrix taiwanensis sp. nov. Head vertex without a longitudinal stripe of dark pigment between eyes, each eyepatch with a posterior dark spot (Fig. 5D), dorsal large abdomen lacking any dark spot, dorsal small abdomen almost entirely covered by a large patch of dark pigment. Ant I red-purple, Ant II basally pale red, apically dark purple, Ant III–IV dark purple. Legs yellowish, banded with three purple stripes on tibiotarsus. Furca pale. Most body chaetae smooth (not denticulate). Head: Antennae shorter than body, 1.27 mm in holotype. Holotype antennal segments ratio of Ant I:II:III:IV as 1:6.73:7.7:1.8. Antennal chaetotaxy as in Ptenothrix taiwanensis sp. nov., with no observable differences between the two species, Ant I with 7 regular chaetae, two of them ventral (as in Fig. 7A), Ant II with 20 regular chaetae plus 3 cup sensilla (as in Fig. 7B), Ant III distally partially divided into about 8 distal pseudo-subsegments, with 80 regular chaetae, 10 cup sensilla and 1 microsensillum, apical organ typical, with 2 sense rods inside a single shallow invagination (as in Fig. 7C), Ant IV with 4 subsegments and about 61 regular chaetae (as in Fig. 7D). Head length (along the longitudinal of the body) 243.5 µm in a female paratype (holotype unclear due to position), head height (from mouth to vertex) 613.97 µm in holotype. Head chaetotaxy mostly similar to Ptenothrix taiwanensis sp. nov. Eyes 8+8 with black pigment, eyepatches with 2 interocular spine-like chaetae, the internal larger than the external one (Fig. 14A). Head frontal area with only A, D, E lines, with 2/3/1 spines, respectively (6+6 spines in total), D line central spines sharp and as long as the lateral ones (Figs. 12B, 14A). Interantennal area α, β and γ lines with 2/2/1 short chaetae, respectively (as in Fig. 8A); clypeal area a–f lines (dorsal and ventral areas combined) with 8(+1)/8(+1)/5/4/4(+1)/5(+1) chaetae respectively; b, c, e and f lines with 2 cup sensilla each. Distal margin of the clypeus with 3 prelabral chaetae, chaetotaxy of labral with 2(+1) p, 2(+1) m + 2 page 19 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan Fig. 14. Ptenothrix matsuensis sp. nov. main chaetotaxic diagnostic features compared to P. taiwanensis sp. nov.: (A) Head interocular, frontal and interantennal chaetotaxy, black arrows highlight the similar lengths of the lateral and internal chaetae of D line; (B) Large abdomen chaetotaxy (partially represented), white arrows mark chaetae/spines present or absent, the black arrow points to a regular chaeta which is blunt acanthoid in P. taiwanensis sp. nov.; (C) Small abdomen of male (labels following Betsch 1997), black arrows point to two chaetae between aai1 and ai6; (D) Manubrium dorsal (posterior) chaetotaxy, with 7+7 chaetae. page 20 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan a chaetae, a2 thicker than the others, p0 and p1 chaetae reduced; labrum without clear formed papillae (as in Fig. 8B). Mandibles asymmetrical with 4–6 apical incisive teeth (left with 6 teeth, right with 4 teeth); maxilla capitulum globular, with 6 lamellae, 3 smooth and 3 fringed, without any clear modification (as in Fig. 8C and D). Labial papilla E with 4 guard chaetae, lateral process finger-shaped, almost reaching the apex of the papilla (as in Fig. 8E). Maxillary outer lobe with 2 chaetae subequal in length, apical one thicker than the basal one, sublobal plate with 2 chaetae-like appendages (as in Fig. 8F). Ventral groove with 1 and 1 surrounding chaetae from lines a and b, respectively, labium basal fields with 4 central (a2 longer than the others) and 5 lateral chaetae (as in Fig. 8G). Trunk (large and small abdomens): Trunk length of holotype 1.31 mm. Large abdomen: thorax continuous with abdomen, without any visible segmentation or constrictions. Chaetotaxy similar between males and females, and almost identical to Ptenothrix taiwanensis sp. nov. Th II with 1 a and 3 to 4 m chaetae, none as large blunt acanthoid chaetae; Th III with 1 a and 3 m chaetae; Abd I with 3 a chaetae; bothriotricha A, B, C present and misaligned, A posteriorly to B–C and on a large papilla with a surrounding cup sensillum, B and C with 4 surrounding chaetae between them, each inside a small papilla; dorso-posterior region with 4 longitudinal lines of spine-like chaetae, with about 5–6/5/12–14/12– 14 chaetae for the more lateral to the more dorsal line, respectively (Fig. 14B). Parafurcal area (furca basis) without neosminthuroid chaetae, with 2 cup sensilla, 2 large blunt chaetae and 7 regular chaetae. Bothriothricum D in the large abdomen, in a small papilla (as in Fig. 9A). Small abdomen of the female: dorsal anal valve with as1–5, ms1–4, mps1–4, mps’ and ps1–2 chaetae, as1, ms1 and ps1 unpaired; as4–5 as cup sensilla, as1–2, ms2, ms4 and mps4 as blunt acanthoid chaetae, ms3 as a S chaeta; ventral anal valves each with aai1, ai1–6, ami1–2, mi1–5, mpi1–2 and pi1–3 chaetae, ami2 as an oval organ; ai1 as a cup sensillum, ai2–3 as S chaetae, aai1 and mi1 as a blunt acanthoid chaetae, mi5 as the subanal appendage, smooth, blunt and thick; 3 chaetae present between aai1 and ai6 (as in Fig. 9B). Small abdomen of male: dorsal anal valve with as1–5, ms1–4, mps1, mps4, mps’, and ps1–2 chaetae, as1, ms1, ps1 unpaired, as4–5 as cup sensilla, as1–2, ms2, ms4, mps4 as blunt acanthoid chaetae, ms3 as S chaeta; ventral anal valves each with aai1, ai1–6, ami1–2, mi1– 5 and pi1–3 chaetae, ai1 as a cup sensillum, ai2–3 as S chaetae, ami2 as an oval organ, mi1 as a blunt acanthoid chaeta; 2 chaetae present between aai1 and ai6 (Figs. 12D, 14C). Genital plates of female and male unclear. Large abdomen appendages: overall chaetotaxy of legs, ventral tube, tenaculum and furca as described to Ptenothrix taiwanensis sp. nov. Epicoxa, subcoxa and coxa I with 0, 0, 1 chaeta, respectively; trochanter I with 4 regular chaetae (as in Fig. 9A); femur I with 12 regular chaetae plus 1 microsensillum in a cavity (oval organ) and 1 cup sensillum (as in Fig. 10A); tibiotarsus I with 40 regular chaetae plus 1 microsensillum in a cavity (oval organ), 3 cup sensilla and 4 oval organs (without inner sensilla), distal whorl with 8 chaetae (as in Fig. 10B). Epicoxa, subcoxa and coxa II with 1, 1, 3 chaetae, respectively (as in Fig. 9A); trochanter II with 5 regular chaetae; femur II with 14 regular chaetae plus 1 microsensillum in a cavity (oval organ) and 1 cup sensillum (as in Fig. 10C); tibiotarsus II with 41 regular chaetae plus 1 microsensillum in a cavity (oval organ), 3 cup sensilla and 4 oval organs (without inner sensilla), distal whorl with 9 chaetae (as in Fig. 10D). Epicoxa, subcoxa and coxa III with 1, 1, 5 chaetae (including one apical small acanthoid chaetae), respectively (as in Fig. 9A); trochanter III with 6 regular chaetae; femur III with 14 regular chaetae plus 1 microsensillum in a cavity (oval organ) and 1 cup sensillum (as in Fig. 10E); tibiotarsus III with 37 regular chaetae plus 2 modified serrated chaetae, 1 microsensillum in a cavity (oval organ), 5cup sensillum, and 4 oval organs (without inner sensilla), distal whorl with 9 chaetae, number of teeth on modified serrated chaetae variable, from 4 to 7 (as in Fig. 10F). Tibiotarsus I lacking any apical bent sinusoid chaeta. Inner apical macrochaeta spiky in tibiotarsus I and blunt in tibiotarsi II–III (as in Fig. 10B, D and F). Foot complexes I–III similar to Ptenothrix taiwanensis sp. nov., with 2 small pretarsal chaetae each, 1 anterior and 1 posterior; ungues with 2 unpaired inner teeth subequal in size, lateral lamellae with 2+2 teeth, duplicate teeth not seen, pseudonychia present, merged to the dorsal face with 2 unpaired dorsal teeth, the proximal one smaller, with lateral margins irregularly serrate. Unguiculi with 1 basal tooth and 1 axial filament surpassing the tip of the ungues, apical filaments acuminate or slightly knobbed (as in Fig. 11A–C). Collophore with 1+1 chaetae on corpus plus 1+1 distal chaetae on lateral flaps (as in Fig. 11D), with a pair of long and warty sacs. Tenaculum with a quadridentate ramus, corpus with 4 apical chaetae. Furcal size length in holotype as manubrium = 153.62 µm; dens = 421.74 µm; and mucro = 149.07 µm. Manubrium with 7 dorsal (posterior) chaetae on each side (Fig. 14D); dens dorsally (posteriorly) with 24 chaetae: 10 on the dorsoexternal (E1–10), 10 on the dorso-internal (J1–10) and 4 dorso-central (PE1–4) chaetae, respectively, external and internal chaetae feathered, except for the chaetae near the manubrium and an apical-external one (as in Fig. 11F); dens ventrally (anteriorly) with 8 chaetae, with the following formula from the apex to the basis: 3, 2, 1, 1… 1; mucro with a narrow apex, with both edges page 21 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan serrated, with about 46 (inner) and 34 (outer) serrations on each edge, with an apical notch. Ratio mucro: dens: manubrium in holotype 1: 2.83: 1.03. Distribution: Nangan Island, Matsu, Taiwan. Habitat: Specimens of Ptenothrix matsuensis sp. nov. were found on dead leaves of tree ferns and Miscanthus bushes, similarly to Ptenothrix taiwanensis sp. nov. Sampling localities are presented as green triangles in figure 1. It is noteworthy that Ptenothrix matsuensis sp. nov. was only found during the wet season in Nangan Island, while no specimens were collected during the dry season, a condition that could be related to the life history of the species. Remarks: Ptenothrix matsuensis sp. nov. is remarkably similar to P. taiwanensis sp. nov. in overall morphology (Fig. 5), but they appeared as distinct taxa in our analyses of COI phylogeny (Fig. 6) and K2P distances (Table 1). Even so, P. matsuensis sp. nov. can be objectively distinguished from P. taiwanensis sp. nov. by: head vertex with an unclear or absent central longitudinal pigment (with a central longitudinal stripe of dark pigment in the latter), frontal head D internal spines as long as the lateral ones (shorter than the lateral ones in P. taiwanensis sp. nov.), manubrium with 7 dorsal chaetae (vs. 8–10 in P. taiwanensis sp. nov.), and only 2 chaetae present between aai1 and ai6 in the small abdomen of the male of P. matsuensis sp. nov. (3 chaetae present in the male of P. taiwanensis sp. nov.) (Fig. 12). For further comparisons between similar species of Ptenothrix from Taiwan, South China and Japan, see table 2. Lectotype designation of Ptenothrix denticulata (Folsom, 1899) The type material of Papirius denticulatus Folsom, 1899 in NMNH is a slide-mounted specimen, and is labeled as “Ptenothrix (Papirius) denticulatus(sic) (Folsom) Type No 5076” and “Komaba, Tokyo, Japan. Coll. C. Ishikawa. Nov. 6. 1894. Lectotype det. by J. T. Salmon, 1938”. Although the specimen is labeled as “lectotype” on the slide, the lectotype designation was never formally published, rendering it an invalid type designation under ICZN Art. 74. In 1938, while planning to write a monograph on Collembola of the world, Salmon visited the museums as part of a tour in various institutions in Europe, Great Britain, and the United States to examine and re-describe poorly described Collembola type specimens. Nevertheless, the monograph was never finished. In 1974, the author published the drawings he had completed in the Zoology Publications from Victoria University of Wellington (Salmon 1974), but Ptenothrix denticulata was not included in this article (Dana M. De Roche, personal communication, January. 12. 2023). As a result, we herein designate the specimen of Papirius denticulatus Folsom, 1899 in the Smithsonian Institution National Museum of Natural History, Washington, USA (catalog no. USNMENT01747048) as the lectotype for Ptenothrix denticulata (Folsom 1899). DISCUSSION Folsom (1899) originally described three specimens of Papirius denticulatus without designating a holotype. In 1938, Salmon examined these specimens and classified them as two different species: Dicyrtoma denticulata (Salmon, 1964:634), with a type specimen deposited in the Museum of Comparative Zoology, Harvard, USA and Ptenothrix denticulata (Salmon, 1964:642), with two type specimens, one deposited in the Museum of Comparative Zoology, Harvard, USA, and the other in the Smithsonian National Museum of Natural History, Washington, USA. Salmon differentiated Dicyrtoma from Ptenothrix based on the presence or absence of annulations on Ant. III and Ant. IV, with Dicyrtoma lacking these distinct annulations and Ptenothrix having them. Subsequent research by Betsch (1980) demonstrated that annulations on Ant. III are ontogenetic, being absent or indistinct in juveniles and becoming distinct in adults. Following Betsch’s findings, we can reasonably conclude that the specimen classified as Dicyrtoma denticulata by Salmon (1964), which is now missing, is a juvenile form of Ptenothrix denticulata. Therefore, all three specimens originally described by Folsom (1899) belong to a single species, Ptenothrix denticulata. Our re-evaluation shows that Dicyrtoma denticulata (Salmon, 1964:634) is synonymous with Ptenothrix denticulata (Folsom, 1899), specifically a juvenile form. Consequently, the specimens should be referred to as Ptenothrix denticulata (Folsom 1899), with Dicyrtoma denticulata (Salmon, 1964:634) as a synonym of Ptenothrix denticulata (Salmon, 1964:642) (syn. nov.). Color patterns and their variations are commonly regarded as useful diagnostic characters that should not be overlooked, especially when there is a constant variation of these features between species of the same genus (Soto-Adames 2002; Ding et al. 2019). As a result, the detailed depiction of color patterns should not be omitted in morphological descriptions, since they are helpful for further taxonomic comparisons. Our finding that specimens previously identified as Ptenothrix denticulata (Folsom, 1899) in Cheng et al. (2022) that have slightly different color patterns actually belong to two new species adds another piece of evidence page 22 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan supporting the importance of carefully studying this feature in different taxa, as well as indicating that the diversity of Collembola in Taiwan is still underexplored (Cheng et al. 2022). In addition to Taiwan and Japan, based on image records curated in http://www. collembola.org, specimens that look superficially similar to the Ptenothrix species investigated in this study have also been found in Massachusetts, USA (Bellinger et al. 1996–2024). While the identities of the imaged animals are unclear, as well as whether they are native or introduced, the presence of a species potentially related to Asiatic taxa on the other side of the Pacific Ocean highlights the need for further research. Accordingly, clarifying the taxonomic identities of Ptenothrix denticulata and Ptenothrix denticulata catenata with redescriptions and molecular data is critical for the taxonomic foundations of Asiatic Ptenothrix. Taxonomic studies of Ptenothrix and other springtails in Taiwan are fundamental and critical for a better understanding of the real species richness of the country, dispersal mechanisms and taxa distribution, all important data which can be used to outline strategies for the conservation of Taiwanese soil fauna. When we surveyed the original descriptions of Ptenothrix species found in Taiwan, South China, and Japan, we found that many taxa only have oversimplified morphological descriptions. Many details of most species are still unknown, such as the chaetotaxy of antennae and legs, and the number and distribution of dorsal spines on the large abdomen. This made it difficult not only to understand the morphologies of these species, but also to look for appropriate characteristics for interspecific comparisons. Hence, more comprehensive descriptions, incorporating molecular information whenever possible, will help to resolve potential taxonomic issues as well as facilitate further integrative studies. CONCLUSIONS Ptenothrix taiwanensis sp. nov. and P. matsuensis sp. nov. are described and illustrated in this study. Morphology of the Ptenothrix species distributed in Taiwan, Japan, and South China are compared. Interspecific and intraspecific genetic distances and phylogenetic analysis are used to support that the two species of Ptenothrix are new to science. In addition, we designate the lectotype of P. denticulata (Folsom, 1899). The discoveries of the present study suggest that further surveys are required to fully understand the diversity of Collembola in Taiwan. Acknowledgments: We are grateful to two anonymous reviewers for their constructive comments on an earler version of this article. We thank Dr. Crystal A. Maier (curator) Whit Farnum (imager) in the Museum of Comparative Zoology at Harvard University, USA, and Dana De Roche, the curator of the Smithsonian National Museum of Natural History, Washington D.C., USA, for providing access to the museum collections. We appreciate Dr. Hui-Yun Tseng in the NTU Insect Museum, Department of Entomology, National Taiwan University, Taiwan, and Dr. Jing-Fu Tsai, Dr. Mei-Ling Chan, and Bao-Cheng Lai at the National Museum of Natural Science, Taiwan for helping archive the specimens newly collected in this study. We also thank Dr. Yu-Feng Hsu for providing helpful advice about ICZN related issues. We are thankful to the following people who provided important samples for this study: Yu-Hsiang Ho, Tzu-Po Lin, Hou-Feng Li, Hsiang-Yun Lin, BingJhih Liou, Wei-tsen Liu, Chieh Kao, Li-Hung Chen, Tsai-Wen Hsu. We are also thankful to the staff of Komabano Park, Meguro-ku, Tokyo, Japan for allowing samples collection in the park. We are grateful to the staff of Technology Commons, College of Life Science, NTU for assistance with scanning electron microscopy. This study was supported by the National Science and Technology Council, Taiwan (MOST 108-2621-B-002001-MY3 and MOST 111-2621-B-002-005-MY3) and the Ministry of Education, Taiwan (Yushan Fellow Program) to C.-H. Chang, the National Science and Technology Council, Taiwan to H.-J. Cheng (1122927-I-002-527), and the Brazilian National Council for Scientific and Technological Development (CNPq), grant number 309114/2021-7, to B.C. Bellini. Authors’ contributions: HJC, FJ, and CHC initiated the study. HJC and TN conducted the sampling. HJC and BCB conducted the drawing and morphological descriptions. HJC and CHC performed molecular analysis and drafted the manuscript. Competing interests: HJC, BCB, FJ, TN and CHC declare they have no conflicts of interest. Availability of data and materials: DNA sequences newly acquired in this study are deposited in GenBank. Specimens are deposited at the NTU Insect Museum, National Taiwan University (NTUE), Taipei, Taiwan and the National Museum of Natural Science (NMNS), Taichung, Taiwan. Consent for publication: Not applicable. Ethics approval consent to participate: Not applicable. page 23 of 25Zoological Studies 63:42 (2024) © 2024 Academia Sinica, Taiwan REFERENCES Arlé R, Mendonça C. 1982. Estudo preliminar das espécies de Dicranocentrus Schött, 1893, ocorrentes no Parque Nacional da Tijuca, Rio de Janeiro (Collembola). Revista Brasileira de Biologia 42:41–49. Bellinger PF, Christiansen KA, Janssens F. 1996–2024. Checklist of the Collembola of the World. 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