﻿Systematic notes on three new Luthela (Mesothelae, Heptathelidae) spiders from China, with their descriptions

﻿Abstract Three new segmented trapdoor spider species belonging to the family Heptathelidae Kishida, 1923, i.e., Luthelaasukasp. nov. (♂♀, Sichuan), L.beijingsp. nov. (♂♀, Beijing), and L.kagamisp. nov. (♂♀, Sichuan), are described from China. Their phylogenetic position and relationships within Heptathelidae are tested and assessed using a combination available COI data downloaded from GenBank with new DNA sequences obtained in this study. The results show that the new species form a clade with eight known and one undescribed species of Luthela. High-definition illustrations of the male palps and female genitalia, diagnoses, and DNA barcodes are provided for these three new species, and their distributions are mapped.


Introduction
Mesotheles, commonly known as primitively segmented spiders, are characterized by having a series of plates on the abdomen and the spinnerets situated in the middle of ventral abdomen. The suborder Mesothelae previously included only one extant family Liphistiidae Thorell, 1869 (s.l.), which has now been split into two closely related families, Heptathelidae Kishida, 1923 andLiphistiidae Thorell, 1869 (s.s.) (Petrunkevitch 1939).
The family Heptathelidae currently consists of 107 extant species in seven genera, whose range is limited to the Far East, such as in Japan, the Ryukyu Islands, China, and Vietnam (Xu et al. 2021;WSC 2023). This family was originally described as a tribe (Heptatheleae) of Liphistiidae (s.l.) by Kishida (1923), and subsequently was elevated to the level of a family by Petrunkevitch (1939) and confirmed by Haupt (1983). Raven (1985) synonymized Heptathelidae with Liphistiidae (s.l.). Recent molecular phylogenetic studies (Xu et al. 2015a(Xu et al. , 2015b(Xu et al. , 2021 have confirmed the monophyly of Liphistiidae (s.l.) as well as that of its two subfamilies, Heptathelinae and Liphistiinae. Li (2022) restored the subfamily Heptathelinae to the family level and circumscribed Liphistidae (s.s.) to include only all extant species of Liphistius Schiødte, 1849. Based on extensive comparisons of the estimated divergence time in extant spider families and known fossils, Breitling (2022) suggested that it would make more sense to reunite both families into Liphistiidae (s.l.). WSC (2023) took note of Breitling's viewpoint, but at present rejected his proposal on the grounds that the age of splitting is not sufficient reason to reunite the families.
Luthela Xu & Li, 2022, an endemic genus of northern China, was newly erected and delimited on the basis of morphological characters and molecular data, and it was transferred from Liphistiidae to Heptathelidae (Li 2022;Xu et al. 2022). At present, Luthela includes eight known extant species, which are distributed almost exclusively north of the Yangtze River to the Yellow river basin in China, but no species have been recorded in Beijing and Sichuan.
The aims of this paper are 1) to describe and illustrate the three new species; 2) to provide the COI sequences of them for verifying their sex pairing; 3) to test their phylogenetic position and relationships within heptathelids; and 4) to map the geographic distributions of these extant Luthela species. This paper expands the knowledge of species diversity of Chinese Heptathelidae.

Specimens sampling
Specimens studied here were collected from Beijing City and Sichuan provinces, China, on 8 October 2019, 15 June 2022, 16 October 2022, and 30 January to 1 February 2023. All specimens were captured by hand and stored in 95% ethanol at −20 °C.

Molecular data
To test the taxonomic position of the three Luthela species, five individuals were selected from the examined materials for molecular data collection. The first and second legs on the right were used to extract genomic DNA and sequence the gene fragments COI. The rest of the bodies were kept as vouchers. All molecular data were obtained from specimens collected at the type localities of the species, although not from the type specimens themselves. Whole genomic DNA was extracted from tissue samples with the Universal Genomic DNA Kit (CWBIO, Beijing, China) following the manufacturer's protocol for animal tissue. The COI gene fragments were amplified in 50 µL reactions. Primer pairs and PCR protocols are given in Table 1. Raw sequences were edited and assembled using Mesquite v. 3.02 (Maddison and Maddison 2011). New sequences were deposited in GenBank (Table 2). All molecular vouchers and examined materials are stored in the Natural History Museum of Sichuan University in Chengdu, China (NHMSU).
To place these new species in a proper taxonomic position within Heptathelidae and verify their sexual pairing, we used these sequences and a selection from previously sequenced taxa to assemble a phylogeny of heptathelid spiders: Ganthela Xu & Kuntner, 2015, Heptathela Kishida, 1923, Luthela, Qiongthela Xu & Kuntner, 2015, Ryuthela Haupt, 1983, Songthela Ono, 2000, and Vinathela Ono, 2000. In addition, a Liphistius species was used as the outgroup (Table 2). Sequences were aligned with MAFFT v. 7.505 (Katoh and Standley 2013) using '-auto' strategy and normal alignment mode. Best partitioning scheme and evolutionary models for three predefined partitions were selected using PartitionFinder2 v. 2.1.1 (Lanfear et al. 2017), with all algorithms and Akaike information criterion (AIC). SYM+I+G, HKY+I+G, and GTR+G were selected for the first, second, and third codon positions of COI, respectively.
Bayesian phylogenetic inference (BI) was performed using MrBayes v. 3.2.7 (Ronquist et al. 2012) through Phylosuite v. 1.2.3 (Zhang et al. 2020) using four Markov Chain Monte Carlo (MCMCs) chains with default heating parameters for 50,000,000 generations or until the average standard deviation of split frequencies was <0.01. Markov chains were sampled every 5000 generations, and the first 25% of sampled trees were burn-in. The website iTOL v. 6.7 (Letunic and Bork 2021) was used to analyse the performance of our BI analyses. Maximum-likelihood (ML) phylogenies were also inferred using IQ-TREE v. 2.0 (Nguyen et al. 2015) through Phylosuite v. 1.2.3 (Zhang et al. 2020) under Edge-linked partition model for 1000 ultrafast (Minh et al. 2013) bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihoodratio test (Guindon et al. 2010).

Morphological data
Specimens were examined and measured with a Leica M205 C stereomicroscope. All male palps and female genitalia were dissected from the bodies before being examined and photographed. To reveal the internal structure, female genitalia were boiled for 5 min in KOH solution (1 mol/L) at 45 °C, and then a dissection needle was used to remove the remaining soft tissue before being photographed. Photographs of male palps and female genitalia were taken with a Canon EOS 60D wide zoom digital camera (8.5 megapixels) mounted on an Olympus BX 43 compound microscope. The

Phylogenetic analysis
The BI analysis of the dataset of COI genes recovered a single parsimonious tree topology. This tree shows heptathelids are monophyletic but with low support. All 29 heptathelid species included are divided into two major clades, and the seven genera they represent formed the following phylogenetic relationships: (Songthela + (Vinathela + (Ganthela)) + (Luthela + (Qiongthela + (Ryuthela + Heptathela)))). These seven genera are also monophyletic, with high support in clades of Songthela, Vinathela, Qiongthela, Ryuthela, and Heptathela, but low support in the Ganthela and Luthela clades. Three new species (Fig. 1, indicated by red font) are nested within Luthela, which is a clade composed of 12 Luthela species (Fig. 1, indicated by a pink box). The sex pairing of all three new species were confirmed to be correct and highly supported as separate clades and belong to the genus Luthela. The sister group relationship of Luthela asuka sp. nov. and Luthela kagami sp. nov. has high support. The same relationship occurs between Luthela beijing sp. nov. and Luthela handan Xu et al., 2022. These results support our taxonomic decision to recognise them as new species and confirm their higher affinities.
The result of ML is consistent with that of the BI on some major clades, but there are some differences (Fig. 2). In the ML tree, all 29 heptathelid species also clustered into a monophyletic group. Different from the topology structure of BI tree, the phylogenetic relationships of the seven genera they represent are as follows: (Vinathela + (Songthela + (Ganthela + (Luthela + (Qiongthela + (Ryuthela + Heptathela)))))). Also, as in the BI tree, the clades of Vinathela, Songthela, Qiongthela, Ryuthela, and Heptathela have high support, but the clades of Ganthela and Luthela have low support. As a sister group, the clade of Luthela is delimited to include eight known, three new, and one still undescribed species. Both BI and ML analyses show that the three new species form a clade which is the sister group to remaining Luthela species. The available molecular evidence supports the taxonomic placement of the three new Luthela species.  Diagnosis. Males of Luthela differ from those of other heptathelid genera except Songthela, by the smooth conductor with one or two long spines (see ASC and BSC in Figs 3C, 4B, 5B, 6B), and they can be distinguished from the males of Songthela in having regular larger teeth on the contrategular margin (see CT in Figs 3B, 3C, 4B, 5D, 6B, 6C). Females of Luthela can be recognized from those of other genera by the middle pair of the receptacular clusters being situated at the anterior margin of the bursa copulatrix and the lateral ones at the dorsolateral position of the bursa copulatrix (Fig. 3H, 5F, 5H, 6H).
Distribution. Northern China, from the Yangtze River to the Yellow river basin. Etymology. The specific epithet is from "Asuka Langley Soryu", a fictional character wearing a red combat suit from the animation "Evangelion" (by the Japanese creator Hideaki Anno), refers to the body color; noun (name) in apposition.

Luthela asuka
Diagnosis. Males can be distinguished from those of congeners, except L. kagami Wei & Lin, sp. nov., in lacking the BSC (Fig. 4A), contrary to other species (cf. Xu et al. 2022: figs 3B, 5E, 6B, 7E, 10B, 12B, 14D), and in having the contrategulum bearning relatively dense, smaller serrated teeth (Fig. 4B, E), rather than sparse and larger teeth in other species (cf. Xu et al. 2022: figs 3A, 5D, 6B, 7D, 10H, 12D, 14H). Males also differ from L. kagami sp. nov. in having two nearly invisible lateral teeth on the middle portion of the conductor and the longer TA ( Fig. 4A-C, E, F), rather than two relatively larger teeth and a shorter TA in the latter (Fig. 7B-D, F). Females differ from those of congeners in having the paired receptacular clusters situated at the relatively short genital stalks and in the relatively smaller size (Fig. 4G, H), rather  than the long genital stalks and the larger size (cf. Xu et al. 2022: figs 4, 5H, I, 6H-M, 8, 9, 11, 13, 14H-M). Females differ from those of L. kagami sp. nov. in having the receptacular clusters relatively separated and the lateral pair larger than the middle pair (Fig. 4G, H), rather than closer and nearly equal in size (Fig. 7G, H).
Palp (Fig. 4A-F): prolateral paracymbium pale, weakly sclerotized, with numerous setae and spines at distal and retrolateral surface. Contrategular margin denticulate, with large teeth on proximal part and smaller but denser teeth on distal part. Marginal apophysis of tegulum serrated, with tapering terminal apophysis of tegulum, margin of dorsal extension of terminal apophysis with teeth varied in size and distance. Conductor smooth, fused to embolic base, with large apical spine and 2 tiny lateral spines on middle portion. Embolus with translucent, flat opening and several ribbed ridges distally.
Female genitalia (Fig. 4G, H). Two pairs of receptacular clusters situated on short and thick stalks; lateral pair relatively larger than middle pair. Middle pair of receptacular clusters separated from each other, situated on anteromargin of bursa copulatrix; lateral receptacular clusters situated slightly dorsolaterally.
Distribution. Known only from the type locality (Fig. 8). Etymology. The specific epithet derives from the type locality; noun in apposition. Diagnosis. Males of this new species can be recognized from those of other congeners, except L. handan, L. schensiensis, L. yiyuan, and L. yuncheng, by the conductor having 2 spines of nearly equal length and by having a lateral tooth on the middle portion of conductor (Fig. 5B, F), rather than 2 spines in unequal length or lacking a lateral tooth on the conductor (cf. Xu et al. 2022  basal spine of conductor thinner and shorter and the promixal part of the margin of the marginal apophysis with a row of smaller teeth (Figs 5B, F, 6B, D), rather than with a thick, long basal spine on the conductor and the proximal margin of the marginal apophysis with 3 larger teeth. (cf. Xu et al. 2022: fig. 7E, G). Females can be distinguished from those of congeners in having the 2 paired receptacular clusters with longer genital stalks and the lateral pair equal to ca 2× size the middle ones (Fig. 6E-H), rather than shorter genital stalks and the lateral receptacular clusters greater than 3× or less than 2× the middle ones in size (cf. Xu et al. 2022: figs 4, 5H, I, 6H-M, 8, 9, 11, 13, 14).
Palp (Figs 5A-F, 6A-D): prolateral paracymbium pale, weakly sclerotized; distal and retrolateral sides with numerous setae and spines. Contrategulum with denticulate margin, with 7 teeth, the fifth bifurcated, and only 4 large teeth visible in dorsal view. Posterior part of marginal apophysis of tegulum serrated, with regular, small denticles; terminal apophysis of tegulum relatively long, apex pointed in distal view, margin of dorsal extension of terminal apophysis with teeth nearly equal in size and distance. Conductor smooth, fused to embolic base, 2 long spines separated at a wide angle, a small tooth located between upper spines and lower spines of conductor. Embolus with translucent, flat opening, and several ribbed ridges distally.
Female genitalia (Fig. 6E-H). Two pairs of receptacular clusters situated on stalks, middle pair of receptacular clusters separated from each other, on anteromargin of bursa copulatrix, distinctly smaller than lateral pair. Lateral receptacular clusters dorsolateral, stalks thick.
Distribution. Known only from the type locality (Fig. 8). Etymology. The specific epithet is from "Hiiragi Kagami", a fictional character from the comic "Lucky Star" (written and illustrated by the Japanese cartoonist Yoshimizu Kagami) with haircut similar to "Asuka Langley Soryu" (see Etymology of Luthela asuka sp. nov.); the name refers to the great similarity between these two new species; noun (name) in apposition.

Luthela kagami
Diagnosis. Males can be distinguished from those of other congeners, except L. asuka sp. nov., in lacking BSC (Fig. 7A), in contrast to other species (cf. Xu et al. 2022: figs 3B, 5E, 6B, 7E, 10B, 12B, 14D), and in the contrategulum having relatively dense, smaller serrated teeth (Fig. 7B, F), rather sparse but larger teeth in other species (cf. Xu et al. 2022: figs 3A, 5D, 6B, 7D, 10H, 12D, 14H). Males differ from those of L. asuka sp. nov. in having two relatively large teeth on the middle portion of conductor and a shorter TA (Fig. 7B-D, F), rather than with two tiny, nearly invisible teeth and a longer TA (Fig. 4A, B, E). Females differ from congeners, except L. asuka sp. nov., in having the paired receptacular clusters with relatively short genital stalks and in their relatively smaller size (Fig. 7G, H), rather than long genital stalks and large size (cf. Xu et al. 2022: figs 4, 5H, I, 6H-M, 8, 9, 11, 13, 14H-M). Females can be distinguished from L. asuka sp. nov. in having the receptacular clusters close and nearly equal in size (Fig. 7G, H), rather than separated and with the lateral pair larger than the middle pair (Fig. 4G, H).
Palp (Fig. 7A-F): prolateral paracymbium pale, weakly sclerotized, with numerous setae and spines at distal and retrolateral sides. Contrategular margin denticulate, with large teeth on proximal part, and smaller but denser teeth distally. Marginal apophysis of tegulum serrated, with relatively short terminal apophysis of tegulum; margin of dorsal extension of terminal apophysis with teeth varied in size and distance. Conductor smooth, fused to embolic base, with large apical spine and 2 small lateral spines on middle portion. Embolus with translucent, flat opening and several ribbed ridges distally.
Distribution. Known only from the type locality (Fig. 8).