Callosa gen. n., a new troglobitic genus from southwest China (Araneae, Linyphiidae)

Abstract A new linyphiid genus Callosa gen. n., with two new species Callosa ciliata sp. n. (♂♀, type species) and Callosa baiseensis sp. n. (♂♀), from southwest China are described. Detailed description of genitalic characters and somatic features is provided, as well as light microscopy and SEM micrographs of each species. Callosa gen. n. was found in caves in Yunnan and Guangxi, and its copulatory organs are similar to those of Bathyphantes and Porrhomma, but differ greatly in details. The monophyly and placement of Callosa gen. n. are supported by the results of molecular analysis.


Introduction
In previous collecting work conducted in caves in southwest China, a considerable number of troglobitic spider species belonging to Nesticidae, Leptonetidae, Telemidae, and Pholcidae were found, but Linyphiidae were seldom encountered. Due to insufficient efforts in taxonomy, no more than 100 linyphiid species have been reported from there, and only one of them was found in caves. Here a new linyphiid genus collected in caves from southwest China is described, whose copulatory organs identify it as a genus of Porrhommini. It has obvious somatic characters of real cave dwellers, indicating its long-term underground evolutionary history. In order to test its placement in Porrhommini suggested by morphological characters, an additional molecular analysis based on newly sequenced DNA data of the two species and sequences available from GenBank was conducted.

Materials and methods
Specimens were studied using a LEICA M205 C stereomicroscope. Further details were examined under a BX51 compound microscope. Copulatory organs were examined after being dissected from the spiders' bodies. Left male palps were used, except as otherwise indicated. Female epigynes and vulvae were removed and treated in warm potassium hydroxide (KOH) water solution before study. All embolic divisions, epigynes and vulvae were photographed after being embedded in gum arabic. Photos were taken with an Olympus c7070 wide zoom digital camera (7.1 megapixels) mounted on an Olympus BX51 compound microscope. Images from multiple focal planes were combined using Helicon Focus (version 3.10) image stacking software. All measurements are given in millimeters. Eye diameters were measured at their widest extent. Leg measurements are shown as: total length (femur, patella, tibia, metatarsus, tarsus). The terminology of copulatory organs follows Saaristo (1995), Tanasevitch (2014).
SEM images were taken using the FEI Quanta 450 at the Institute of Zoology, Chinese Academy of Sciences. Specimens for SEM examination were critical point dried and sputter coated with gold-palladium. Specimens were mounted on copper pedestals using double-sided adhesive tape.
The tibial spine formula, which expresses the number of dorsal tibial spines on each of legs I to IV, is given for species in which it differs from the type species of the genus. The patellar spine formula is given only if it differs from the most common one (1-1-1-1).
All type specimens are deposited in the Institute of Zoology, Chinese Academy of Sciences in Beijing (IZCAS), except as otherwise indicated.
Abbreviations used in the text and figures are given below. References to figures in cited papers are noted in lowercase type ( fig.).

Phylogenetic analysis
Analysis conducted here is partially based on the data matrix of Arnedo et al. (2009). A few taxa were taken out, and more taxa of Linyphiinae downloaded from GenBank were added to reconstruct phylogeny. A total of 66 taxa were included for the final test. Partial fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI), 16SrRNA (16S) and the nuclear genes Histone 3 (H3), 18SrRNA (18S) were amplified and sequenced for Callosa ciliata sp. n. and C. baiseensis sp. n. following the procedure in Arnedo et al. (2009). Sequences for each gene were edited in Bioedit (Hall 1999), and aligned in MAFFT (http//mafft.cbrc.jp/alignment/server/). Bayesian inference was performed in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using parameters selected by jModelTest (Posada 2008). The Markov chains were sampled every 1000 generations for 2 million generations, with the first 25% of sampled trees discarded as burn-in. Taxonomic and sequence information of the used taxa are presented in Table 1.
Bayesian inference based on four genes yielded a similar phylogenetic tree to Arnedo's (Arnedo et al. 2009) and Sun's (Sun et al. 2014). The Callosa gen. n. species belong to Porrhommini as indicated by the cladogram (Fig. 10).
Male palp: femur about four times longer than patella; tibia with two trichobothria, one ventral and one retrolateral (Fig. 5B). Cymbium spindle-shaped at dorsal view ( Etymology. This specific name is taken from the Latin word 'ciliatus', meaning 'with cilia', which refers to the median membrane with cilia; adjective.   Diagnosis. It is characterised by the subdivided tip of distal suprategular apophysis (Fig. 1D) and in having three coils in copulatory furrows in epigyne (Fig. 3C). Callosa ciliata sp. n. also has a narrower atrium and shorter parmula.
Remarks. To confirm the species delimitation, the p-distance of COI sequences of C. baiseensis sp. n. and C. ciliata sp. n. was calculated using MEGA 6 (Tamura et al. 2013), and the result is 0.12, which falls within the genetic distance interval of 0.07 to 0.16 among Bathyphantes species and 0.07 to 0.17 in Porrhomma based on data from NCBI (The National Center for Biotechnology Information https://www.ncbi. nlm.nih.gov/).

Discussion
Linyphiidae Blackwall, 1859 is not commonly found in caves. In China, in contrast to more than 370 terrestrial linyphiids, only two species have been reported from caves so far (Song and Li 2009), but none of them exhibited traits of cave adaptation, such as depigmentation, reduction or complete loss of eyes, or elongation of legs (Sket 2008). Callosa gen. n. is the first true troglobiont linyphiid genus discovered in southwest China, encompassing two new species found in caves almost 600 kilometers apart, and they display apparent characters of true cave dwellers. It is assumed their ancestors were widely distributed in the montane area in southwest China, and almost certainly extrinsic forces (e.g. geological events, climatic changes) drove them to colonize the caves, which are considered to be a relatively stable environment.
Callosa gen. n. belongs to Porrhommini as suggested by both molecular analysis (Fig. 10) and morphological characteristics. It is obviously monophyletic, and its distinctive traits in both body and copulatory organs might be a result of long-term solitary evolution. Despite its morphological similarities to Bathyphantes (especially B. approximatus), Callosa gen. n. is situated relatively farther from Bathyphantes in the cladogram (Fig. 10). The taxonomical history of Bathyphantes is long and complicated, and several of its subgenera have now been validated as separate genera (e.g. Kaestneria, Diplostyla, Pacifiphantes) based on the conformation of copulatory organs, and some related genera were also established with species transferred from Bathyphantes (e.g. Cresmatoneta Simon, 1929, Microbathypahntes Helsdingen, 1985. A better-sampled phylogenetic analysis of Porrhommini was presented by Wang et al. (2015), in which Bathyphantes appeared as polyphyletic, with Pacifiphantes zakharovi Eskov & Marusik, 1994grouped with Bathyphantes eumenis (L. Koch, 1879. The split between Porrhomma + Diplostyla and Bathyphantes is not well supported. A similar relationship is recovered in our analysis, where Pacifiphantes zakharovi is clustered with Bathyphantes floralis Tu & Li, 2006 (Fig. 10). It also has been previously pointed out that Pacifiphantes magnificus (Chamberlin & Ivie, 1943) could be a misplacement, and probably grouped with Porrhomma + Diplostyla as indicated by both morphology and DNA barcoding (Slowik and Blagoev 2012). As the type species, Pacifiphantes zakharovi was identified with a super short embolus (Eskov and Marusik 1994: fig. 42), the unique trait supposedly distinguishing it from other similar Bathyphantes, however, the discrepancy between morphology and molecular analysis results demands a more comprehensive analysis on the delimitation of Bathyphantes and its close relatives. . "Micronetines-erigonines" clade is presented in green, the "distal erigonines" clade is colored in orange. Taxa with sequences downloaded from NCBI are listed at the end of each branch in black accordingly, and Callosa gen. n. species are marked in red.