Xiangxi Tujia and Miao Autonomous Prefecture of Hunan Province (approximately 109°10' to 110°22.5'E, 27°44.5' to 29°38'N, hereafter referred to as Xiangxi Prefecture) is situated on the eastern edge of the Yunnan-Guizhou Plateau, hinterland of the Wuling Mountains. It lies at the intersection of three major zoogeographical regions (Central China, South China, and Southwest China) within the upper Oriental boundary of China’s zoogeographical division, covering an area of approximately 15,000 km². The region is characterized by a subtropical monsoon humid climate of suitable temperatures, abundant rainfall, dense river networks, widespread soluble carbonate rocks, well-developed karstic landforms, and numerous karstic caves (Wu 2002). These conditions provide favorable habitats for the development of cave biodiversity (Liu 2021; Tian 2023).

Five white horsehair worms were originally discovered by Professor Zhi-Xiao Liu from Jinji Cave in Yongshun County on 6 October 2014, but the specimens were lost. White horsehair worms were rediscovered in 2019, and our systematic investigation of them began in November 2022 (Table 1). A total of 13 female and 19 male free-living hairworms were collected from six locations in the karstic caves within the Xiangxi Prefecture. During the fieldwork, we focused on examining water pits and their surroundings inside caves, carefully turning over stones. All living worms were placed in water bottles filled with a small amount of water and transported back to the laboratory for further study. The worms were placed in a dark box designed to simulate the cave environment, with the temperature maintained at approximately 15 °C. Water from the cave was changed every five days. Insect hosts infected with horsehair worms were hand-collected from the caves. To find out whether a host was infected with worms, the posterior region was examined, and then the worms were collected by immersing the host in water or by host dissection (Fig. 7D, E). The infected host and the harbored worms were preserved in 95% ethanol for sequencing. Some of the samples were preserved in a solution of 75% ethanol for morphological examination. The specimens are deposited in the Hunan Wuling Mountain Biological Science Museum.

Body length and maximum diameter of the worms were measured using a ruler and a vernier caliper. Morphological characteristics and behavioral habits were observed visually. Fragments (approximately 0.5 cm in length) of the anterior end, mid-body, and posterior end of the preserved samples were examined and photographed using a stereomicroscope (LEICA M205 C). Representative specimens were selected for scanning electron microscopy (SEM) analysis. They were dehydrated using a series of ethanol and acetone solutions (95% and 100% ethanol (twice) and ethanol/acetone mixtures of 2:1, 1:1, 1:2, and 0:1), dried with a critical point dryer, coated by being sputtered with gold, and examined using a Zeiss Field Emission Scanning Electron Microscope (Sigma HD) at magnifications ranging from 100× to 15,000×.

Genomic DNA from adult horsehair worms was extracted using an Animal Genome DNA Extraction Kit (Tsingke, Changsha, Hunan). The partial cytochrome c oxidase subunit I (COI) sequence was amplified using universal primers (LCO1490 and HCO2198) (Folmer et al. 1994). PCR for both primer sets was initiated at 95 °C for 5 min, and amplification was conducted for 35 cycles of 95 °C for 1 min, 50 °C for 1 min, and 73 °C for 1 min, with a final extension at 73 °C for 5 min. We obtained high-quality COI sequences (>500 base pairs) from free-living individuals to be used in our phylogenetic analysis and estimates of intraspecific genetic distances.

The COI gene sequences obtained in this study for horsehair worms were submitted to the GenBank database for sequence alignment. Phylogenetic trees and genetic distance matrices were constructed using the maximum likelihood method implemented in MEGA11 (see detailed information in Table 2). We constructed a maximum likelihood phylogeny using IQ-TREE v2.2.0. First, sequences were aligned with MAFFT using the ‘--auto’ strategy and normal alignment mode. Then, gap sites were removed with TRIMAl v1.2 using the “-automated1” command. Maximum likelihood phylogenies were inferred using IQ-TREE v under the TVM+R3+F model for 50,000 ultrafast bootstraps, as well as the Shimodaira–Hasegawa–like approximate likelihood-ratio test. Additionally, an uncorrected p-distance of COI was conducted in MEGA v.11.0.13.

To date, no specimens of Gordius wulingensis have been found outside the cave in Wuling Mountains. The white color of the specimens suggests an adaptation to living in karstic caves, but as there are other white or lightly colored species living outside of caves (see below), we hesitate to call this species truly cavernicolous. We will continue to investigate the distribution of hairworm in this area in the future, inside and outside of the caves.

The body color of free-living horsehair worms can vary from nearly white to dark brown (Schmidt-Rhaesa 2010). The juvenile, parasitic stages of hair worms are characterized by a white appearance, which gradually turns fully brown after 30 days (in Paragordius varius, Schmidt-Rhaesa 2005). Specimens of Gordius wulingensis described in this study are entirely white. Some other species also exhibit a predominantly white coloration, including Gordius difficilis (white in females and yellowish-brown in males), Gordius terrestris, and Gordius villoti, which ranges from white to dark brown but are actually mainly brown (Valvassori et al. 1988; Bolek and Coggins 2002; Anaya et al. 2019). The cuticle around the cloacal opening and the postcloacal crescent in Gordius wulingensis are pigmented browner than the surrounding cuticle. Gordius paranensis exhibits a brown body coloration with a darker crescent around the cloacal opening compared to the rest of its body (Schmidt-Rhaesa et al. 2000). Gordius chiashanus lacks such color differentiation (Chiu et al. 2020). This suggests that only certain species within the genus Gordius exhibit such color differences.

Although all species of the genus Gordius appear to possess “collar” and “cap” structures at the anterior end, the dark-brown collar may blend into the dark-brown body color, making the boundary between the “collar” and “cap” unclear (Schmidt-Rhaesa 2010).

In Gordius wulingensis, bristles are concentrated at the tips of the inner lobes. Some species in the genus Gordius have such concentrations of bristles in this position; for example, in G. helveticus from Switzerland, bristles are concentrated in a dense patch posterior to the tips of the postcloacal crescent (Schmidt-Rhaesa 2010), while G. karwendeli from Germany and G. terrestris from the United States both exhibit a long row of bristles concentrated medially (Schmidt-Rhaesa 2010; Anaya et al. 2019). Gordius chiashanus has densely distributed bristles on the top and inside of the tail leaf tip (Chiu et al. 2020).

Areoles on the inner wall of the male cloacal opening were first reported in an unidentified species of the genus Gordius (Schmidt-Rhaesa 2013). Subsequently, pebble-like areoles on the inner wall of the male cloacal opening were observed in G. chiashanus (Chiu et al. 2020). The cloacal areoles of Gordius wulingensis are honeycomb-like, differing from the areoles of the aforementioned species.

Gordius wulingensis is also distinguished by longitudinal ridges on both the ventral and dorsal surfaces, a morphological feature absent in other Gordius species.

According to the available COI gene data for horsehair worms presented in the genetic distance table, the closest relatives identified were Gordius sp. 7 (KW-2011-A), Gordius sp. 6 (KW-2011-B), and Gordius sp. 8 (KW-2011-D) from Japan, with genetic distances from 0.162 to 0.190. Gordius sp. 5 (N297B) is distributed in New Mexico, USA, with a genetic distance ranging from 0.236 (Table 3). Due to the lack of specific taxonomic information for this unidentified species, morphological comparisons are challenging (Sato et al. 2012; Hanelt et al. 2015).

In the phylogenetic tree, Gordius wulingensis shares a sister-group relationship with G. paranensis, with a genetic distance ranging from 0.216 to 0.232. Gordius paranensis is distributed in New Zealand and South America, but the specimens included in this analysis (clade 1 and 2) are from New Zealand (Tobias et al. 2017). Gordius paranensis is a very characteristic species with a semicircular row of bristles anterior to the cloacal opening, a character not present in G. wulingensis. In summary, the specimens probably most closely related to G. wulingensis span a wide geographic area and do not shed much light on ancient distribution routes.

Gordius wulingensis can be found year-round within the caves investigated by us. Free-living adults are present in small numbers throughout the year but are more frequently encountered from October to December and July to August. The typical lifespan of free-living adult horsehair worms is 2 to 8 weeks (Bolek and Coggins 2002), but Gordius wulingensis has survived for over three months under laboratory conditions. These findings suggest that, in undisturbed natural karstic caves, adult horsehair worms may survive for more than three months.