First records and a new genus of comb-tailed spiders (Araneae: Hahniidae) from Thailand with comments on the six-eyed species of this family

The family Hahniidae is reported from Thailand for the first time. The genus Hexamatia gen. nov. and two new species, Hexamatia seekhaow gen. et sp. nov. and Hahnia ngai sp. nov., are described and illustrated. DNA sequences are provided for all the species reported here. The phylogenetic position of the novel genus Hexamatia gen. nov. and its relation to Hahnia are discussed. Based on these results, a new combination is proposed for Hexamatia senaria (Zhang, Li & Zheng, 2011) gen. et comb. nov. = Hahnia senaria. Known distribution of the species Hahnia saccata Zhang, Li & Zheng, 2011, originally described from China, is expanded. A brief review and notes on the taxonomy of the six-eyed hahniids are included.


Introduction
The family Hahniidae Bertkau, 1878 is relatively easy to identify due to the advanced location of the tracheal spiracle in relation to the spinnerets and the characteristic arrangement of these in more or less one transverse row (at least in the Hahniinae Bertkau, 1878) (Lehtinen 1967;Opell & Beatty 1976). Other members of this family (e.g., Cicurina Menge 1871 and Cybaeolinae Lehtinen, 1967) do not share this transverse arrangement of the spinnerets (Roth 1967;Wang et al. 2019). The Hahniidae currently includes 351 species in 23 genera distributed worldwide (WSC 2020). The family status of Hahniidae has been confi rmed by molecular phylogenies being placed within the RTA clade, closely related to Cybaeide Banks, 1892 and Dictynidae O. Pickard-Cambridge, 1871 (J.A. Wheeler et al. 2017). However, the relations and delimitations of its genera have always been problematic. Only a few local revisions have been done, two for Nearctic species (Gertsch 1934;Opell & Beatty 1976) and one for New Zealand species (Forster 1970). Beside these revisions, Lehtinen (1967) published some comparative tables including diagnostic characters of 17 extant genera (10 currently valid, WSC 2020) and one more from Baltic amber. Presently, two genera, Cicurina and Hahnia C.L. Koch, 1841, have served as 'wastebin taxa' for new species descriptions, having a great morphological heterogeneity and accounting together for almost 70% of all the valid hahniid species (WSC 2020). The great heterogeneity and unclear delimitations in these and other hahniid genera are a recurrent note in new species publications (Forster 1970;Huang et al. 2017).
The Hahniidae have a worldwide distribution, being more diverse in the Americas and Asia but also having a fair number of species described from Europe, Africa and Oceania (WSC 2020). In Asia, eight genera and 93 species have been recorded distributed from the Middle East to Eastern Russia and Japan. In South and Southeast Asia, hahniids have been reported from Hong Kong, Indonesia, Laos, Philippines, Southern China, Sri Lanka, Taiwan and Vietnam (Lehtinen 1967;Bosmans 1992; Barrion & Litsinger 1995;Tang et al. 1996;Zhang et al. 2011Liu et al. 2015;Huang et al. 2017). This is the fi rst time the Hahniidae are reported from Thailand. Here we describe a new genus and two new species in this family based on molecular and morphological data. Additionally, we include a brief literature review on the rare six-eyed hahniids.

Material and methods
The hahniid species reported here were collected in the Chiang Mai Province, Thailand, between July 16 th and 28 th 2018. All the specimens were captured using methods optimized for ground dwelling spiders: leaf litter sifting, Winkler extractors, pitfall traps and direct collecting on ground, among leaf litter and under rocks or logs.
Specimen habitus and other somatic characters were photographed under a Leica MI6SC stereo microscope equipped with a Nikon DS-Ri2 camera. Genitals were photographed using a Leica DM 2500 microscope attached to the same camera. Specimens were observed in ethanol using semi permanent slide preparations (Coddington 1983). Female genitalia were dissected, digested using pancreatine solution (Álvarez-Padilla & Hormiga 2007) and cleared with methyl salicylate.
Four legs were taken from one individual of each species for DNA extraction. Six gene fragments (COI, H3, 12S, 16S, 18S and 28S) were amplifi ed following M.A.  and Wheeler et al. (2017) protocols; list of primers is provided in the Supplementary material (Supplementary fi le 1). Sequences were edited in Geneious Prime 2020.0.5. New sequences generated for this study were deposited in GenBank; accession numbers are reported in Table 1. All the specimens used here have been deposited in the collection of the Naturalis Biodiversity Center, Leiden, the Netherlands (RMNH. ARA.18411-RMNH.ARA.18415).
We used sequences from the three species we collected, as well as 15 other species with available sequences in GenBank. We used in total 14 species of Hahniidae, three species of Cybaeidae Banks, 1892, and one species of Agelenidae C.L. Koch, 1837, Agelena labyrinthica Walckenaer, 1805, as an outgroup. The sequences used to test the relationships and position of the new species within the Hahniidae are listed in Table 1. We used MAFFT ver. 7.450 online (https://mafft.cbrc.jp/alignment/server/) with default parameters to build the alignments. Alignments for 18S were further trimmed manually due to the size difference of some sequences. 16S and 12S were not used due to the low availability of these loci for the Hahniidae in GenBank; Table 1 only reports accession numbers of these markers for our sequences. Hahnia pusilla C.L. Koch, 1841, type species of Hahniidae, as well as two more species of Hahnia and two of Iberina Simon, 1881 had only COI sequences available in GenBank, therefore, they were not used in our fi nal dataset. Matrix was built using COI, H3, 18S and 28S alignments in Sequence Matrix ver. 1.8 (http://www.ggvaidya.com/taxondna/); matrix is available in Supplementary fi le 2. Each locus was treated as a partition and examined with jModelTest2 (Darriba et al. 2012) in CIPRES (M.A.  to get the best model fi t for each; GTR + I + G was selected in all the cases. Our datasets were analyzed using MEGA X (Kumar et al. 2018) for maximum parsimony (SPR, default values, bootstrap = 1000), RaXML (Stamatakis 2014) in CIPRES for maximum likelihood (GTR, bootstrap = 1000) and MrBayes ver. 3.2.6 (Ronquist & Huelsenbeck 2003) for windows for the Bayesian inference (GTR + I + G, two independent runs with one cold and three heated chains, mcmc =1 000 000 gen, samplefreq = 1000, burnin = 2500). The program Tracer ver. 1.7.1 (Rambaut et al. 2018) was used to analyze the performance of our BI analyses, and Mega X to estimate the genetic distances (JC model, gamma dist., gamma parameter = 1.00; gaps data treatment = pairwise deletion) for our whole dataset.

Abbreviations (in text and fi gures)
A = epigynal atrium ALE = anterior lateral eyes ALS = anterior lateral spinnerets AME = anterior median eyes BI = Bayesian inference Cd = copulatory duct

Phylogenetic analyses
Topologies inferred by the three different phylogenetic analyses recovered nearly identical topologies ( Fig. 1a-c). The genus Hahnia was homogeneously recovered as diphyletic. The clade Hahnia 1 was formed by six species of Hahnia, and Hahnia 2 by H. ngai sp. nov. and H. saccata, the two species of Hahnia we captured in Thailand. The clade Hahnia 1 showed high support, although the internal relationships are not fully resolved, having moderate to weak support values in the ML and MP analyses. This clade was found as a sister group to the new genus Hexamatia gen. nov. in all our trees. The clade Hahnia 2 appears to be more related to Antistea + Neoantistea. This branch is recovered and highly supported in all the analyses. The cluster formed by Antistea + Neoantistea is strongly supported although its internal relationships are not resolved and show weak to moderate support in the MP and ML. The three cybaeid representatives form a highly supported group that is consistently recovered as a sister to the monophyletic Hahniidae. Our BI showed an average deviation of split frequencies below 0.003 after 1 000 000 generations. None of the Estimated Sample size parameters fell below the commonly used threshold of 200 suggesting that our BI ran for an adequate length (Drummond et al. 2006;Lanfear et al. 2016

Diagnosis
Hexamatia gen. nov. is distinguished from most hahniid genera by the combination of the following characters: presence of only six eyes, small body size close to 1 mm, and body pale yellow to white, lacking abdominal patterns in males and having faint chevron lines in females (Zhang et al. 2011: fi g. 23a-b). It can be separated from other six-eyed hahniids by the following combination of characters:

Etymology
The genus name is formed from two Greek roots: hexa (six) and mati (eye). It refers to the number of eyes present in this genus, one of its diagnostic characters. The gender is feminine.

Etymology
The species epithet is a derivation of the Thai seekhaow (white); refers to the lack of color on the body of the holotype of this species.

Distribution
Known from the type locality, Doi Suthep National Park, Chiang Mai, Thailand (Fig. 8).

Notes
See the Discussion for remarks on six-eyed species.

Etymology
The species epithet is a derivation of the Thai ngai (simple), in reference to the relatively simple vulva without the well-formed secondary spermathecae commonly seen in other species of Hahnia.

Discussion
The Hahniidae, especially the Hahniinae, have traditionally been seen as an easily diagnosable group in part due to the transversal comb-shaped position of the spinnerets. Nevertheless, their position as a family has changed overtime, being initially considered a subfamily of the Agelenidae (Simon 1875;  Gertsch 1934;Lehtinen 1967, among others) and Dictynidae (Lehtinen 1967;Paquin & Dupérré 2009;Wang et al. 2019, among others). Currently, the monophyly of the family is largely recognized, and its relations have been indirectly tested as a part of broad scoped phylogenetic studies (J.A. Wheeler et al. 2017). However, the relations between its genera have never been phylogenetically tested. Although our data did not include representatives of all the known hahniid genera, we found some consistent and well supported results with the 14 hahniid species and four loci we analyzed. The position of the new genus Hexamatia gen. nov. as a sister group to the core species of Hahnia in our study is confi dently recovered in all our topologies. We consider that this plus the  morphological differences between the new genus and Hahnia (presence of six eyes, small size close to 1 mm and almost complete lack of coloration and abdominal patterns) are suffi cient to consider it outside of the Hahnia 1 group, and as a genus of its own. We also propose a new combination for Hexamatia senaria gen. nov. Although we were not able to test the relationships between Hexamatia gen. nov. and other six-eyed hahniids like Amaloxenops (Lehtinen 1967;Schiapelli & Gerschman de P. 1958), Intihuatana antarctica (Simon, 1902) (Dupérré & Harms 2018) and Scotospilus , clear morphological differences could be observed in somatic and genital characters like body size, coloration, size and shape of RTA and PA, and the presence of MA (see the diagnosis of Hexamatia gen. nov.).
The clade Hahnia 2 formed by H. saccata and H. ngai sp. nov. was found to be closely related to Antistea + Neoantistea in our analyses (Fig. 1a-c), suggesting that these species might be misplaced in Hahnia. However, these and many other Asian hahniids require a broader revision and more comprehensive phylogeny to fully resolve their relations within this family. Therefore, H. ngai sp. nov. and H. saccata remain in Hahnia; in the case of the later, as it was originally described by Zhang et al. (2011).

Eye reduction in the Hahniidae
This phenomenon appears to be rare in hahniid spiders. Most known species of this family have eight eyes; still, some instances of eye reduction have been documented in at least six genera. The modifi cations of eyes range from size reduction of AME and lack of AME, to complete absence of eyes (Lehtinen 1967). The evolution of this phenomenon in this family has never been studied, and the relations of the species with reduced eyes are largely unknown. Even their taxonomy has been constantly a subject of debate (Lehtinen 1967;Schiapelli & Gerschman de P. 1959;Catley 1999;Dupérré & Harms 2018).
Size reduction of the AME (Fig. 7a) is relatively common being observed in several species of the following genera: Alistra Thorell, 1894 (Lehtinen 1967;Forster 1970;Ledoux 2004), Amaloxenops (Schiapelli & Gerschman de P. 1959;Catley 1999;Dupérré & Harms 2018), Hahnia (Lehtinen 1967;Ubick et al. 2005, among others) and Neohahnia Mello-Leitão, 1917(Mello-Leitão 1917Lehtinen 1967;Heimer & Müller 1988). Reduction in number of eyes ( Fig. 7b-d) is much rarer being documented only in a few species: Amaloxenops vianai Schiapelli & Gerschman, 1958(Schiapelli & Gerschman de P. 1958Lehtinen 1967), Hexamatia senaria gen. nov. (Zhang et al. 2011), Hexamatia seekhaow gen. et sp. nov., Intihuatana antarctica (Dupérré & Harms 2018), Scotospilus longus Zhang, Li & Pham, 2013, and two unpublished species documented in a revision of South American hahniids (Catley 1999); a quick examination of the illustrations and descriptions of these species suggest that they are not closely related. Finally, complete lack of eyes ( Fig. 7e-f) has only been reported in the genus Iberina (Fernández-Pérez et al. 2014;Ledoux 2014). This wide range in the degree of eye reduction and broad geographical spread of this phenomenon suggest that eyes are a very plastic character and the loss or reduction might have evolved independently several times within this family. Nevertheless, a more comprehensive phylogeny of the Hahniidae is necessary to test this hypothesis.