﻿Description and phylogenetic analysis of two new Episinus (Araneae, Theridiidae) species from China

﻿Abstract The spider genus Episinus Walckenaer, 1809 currently contains 66 species worldwide, mostly in warm temperate to tropical areas. This paper describes two new Chinese Episinus species: E.ornithorrhynchussp. nov. (♂♀) and E.papilionaceoussp. nov. (♀). We add these two new and one known Episinus species to the phylogenetic data matrix of Liu et al. 2016 and reanalyze the data. The new phylogeny recovers the monophyly of Episinus and supports its division into two groups, a finding also supported by morphology.

At present, the genus Episinus has 66 described species and of which only 10 have been reported in China (World Spider Catalog 2022). More than half of these species are widespread and also recorded in the Nearctic, Japan, Europe, and Africa (Levi 1955;Yoshida 1985;Okuma 1994;Zhu 1998;Song et al. 1999). Episinus differs from other genera of the Spintharinae by the straight shape of the opisthosoma in lateral view, and in dorsal view with median or posterior humps (Durán-Barron et al. 2013;Rodrigues et al. 2022). Rodrigues et al. 2022 provided a phylogenetic framework based on a morphological analysis, which suggested that Janula and some species of Episinus belong to the same group, and the genus Episinus appeared polyphyletic. The genus Moneta is similar to Episinus, but according to Okuma (1994) can be separated by the alignment of the eyes, the length ratio of the metatarsus to the tarsus, and the structure of the male palp. Our phylogenetic results clearly differentiate between these genera and strongly support the monophyly of the genus Episinus (Fig. 6).
We describe herein two new Episinus species from China, E. ornithorrhynchus sp. nov. and E. papilionaceous sp. nov. We add these species, as well as E. nubilus Yaginuma, 1960, to the data matrix of Liu et al. (2016) and rerun phylogenetic analyses.

Materials and methods
All specimens were kept in absolute ethanol and examined with an Olympus SZX7 stereomicroscope; details were further investigated with an Olympus BX51 compound microscope. Male palps and female genitalia were examined and photographed after dissection from the spider bodies, epigynes were cleared with Proteinase K, and palps were studied after immersion in KOH; habitus photos were obtained using a Leica 205C digital microscope. Left palps are illustrated. All specimens are deposited at the Centre for Behavioural Ecology and Evolution, College of Life Sciences, Hubei University, Wuhan, China (CBEE).
Leg measurements are shown as total length (femur, patella, tibia, metatarsus, tarsus). The number of spines is listed for each segment in the following order: prolateral, dorsal, retrolateral, and ventral (in femora and patellae ventral spines are absent, and the fourth digit is omitted in the spination formula). The terminology used in the text, figure legends, and palp homologies follow Agnarsson (2004) and Agnarsson et al. (2007). All measurements are given in millimeters.

Molecular data
We used the dataset from Liu et al. (2016) and added to it E. ornithorrhynchus sp. nov. and E. papilionaceous sp. nov., as well as E. nubilus Yaginuma, 1960. The final dataset includes 62 theridiid genera and two outgroups representing Nesticidae and Synotaxidae. Sequences for two mitochondrial genes: cytochrome c oxidase subunit I (COI) and ribosomal RNA16S (16S), and three nuclear genes, ribosomal RNAs 18S (18S) and 28S (28S) and histone (H3), were collected for the newly added species. The phylogenetic data collection and analytical methods used conform to Liu et al. (2016).
The appropriate models for the Bayesian analysis were selected with jModelTest2 on XSEDE (2.1.6) (Darriba et al. 2012) using the Akaike information criterion (AIC) (Posada 2008). Bayesian analysis for morphology was carried out using MrBayes 3.2.7a on XSEDE (Huelsenbeck and Ronquist 2001) and maximum likelihood with IQ-Tree stable release 1.6.12 (Chernomor et al. 2016;Nguyen et al. 2015) of individual gene trees as well as concatenated matrices. All large analyses were run in parallel on the CIPRES cluster at the San Diego Supercomputing Center (Miller et al. 2010).

Diagnosis.
Males are similar to E. baoshanensis Liu et al. (2019) but can be distinguished from them by the palpal structure: 1) embolus extends along the left lower lateral aspect of the genital bulb to the posterior margins, but the embolus mainly encircles the left upper lateral and anterior margins of the bulb in E. baoshanensis (Figs 2C,D,3A,B); 2) conductor with a sharp tip in ventral view and not bifurcated, but conductor sclerotized and tip bifurcated in E. baoshanensis (Figs 2C,3A). Females are similar to E. nubilus but can be distinguished from them by the direction of the copulatory duct: fertilization ducts are slightly shorter in the new species, but long and C-shaped in E. nubilus (Fig. 1D). Etymology. The specific name is derived from the Latin adjective ornithorrhynchus, meaning bird's beak, referring to the shape of the conductor; adjective.
Palp (Figs 2C-E, 3A, B). Subtegulum mostly covered by tegulum. Embolus originates at center of palp, a slightly triangular basement that curves upward clockwise. Conductor sclerotized, with a sharp tip extending over apex of cymbium.
Variation. Total length male 3.75-3.79 (n = 2), female 4.10-4.25 (n = 4). Distribution. China (Yunnan Province) (Fig. 5). Diagnosis. This new species is similar to E. xiushanicus Zhu, 1998 in having a peach-shaped structure at the posterior part of the epigynal field but can be distinguished from them by the following characteristics: 1) abdomen without spinous process in ventral view, but the ventral protuberance is spinous in E. xiushanicus (Fig. 4A); 2) spermathecae slightly S-shaped in the new species but W-shaped in E. xiushanicus (Fig. 4D).

Episinus papilionaceous
Etymology. The specific name is derived from the Latin adjective papilionaceous, meaning butterfly-shaped, referring to the shape of the epigynum; adjective.
From the results (Fig. 6), the Latrodectinae is the earliest branching subfamily. Pholcommatinae is polyphyletic and redefined by removing Styposis and Phoroncidia that must be placed in insertae sedis until further data become available. Argyrodinae is in the same monophyletic group as the Phoroncidia, and these are sisters to Spintharinae and Hadrotarsinae. Our results strongly support the sister relationship between Spintharinae and Hadrotarsinae, two specialized groups with most of their species either building reduced webs or having abandoned web building and hunting ants as prey. Anelosiminae is recovered as a monophyletic sister to Theridiinae. Our results strongly support the monophyly of the subfamily Spintharinae. We have also found compelling evidence to support the monophyly of the genus Episinus, although this is weak due to the lack of data for the most-related genera: Janula, Neopisinus, and Brunepisinus. Regardless, we found evidence for the first time for the division of the genus Episinus into two groups. The "angulatus" group to which E. ornithorrhynchus sp. nov., belongs is characterized by the male embolus, and the "nubilus" group to which E. papilionaceous sp. nov. belongs is characterized by female atria. These two groups can be clearly diagnosed based on the following morphological traits: in the "angulatus" group, the male embolus originates at the center of the palp and is close to the cymbium; in the "nubilus" group, female atria are separated by a medium septum. Episinus nubilus Yaginuma, 1960, here newly added to the phylogenetic matrix, was found to be closely related to E. ornithorrhynchus sp. nov. Our findings suggest that the genus Moneta O. Pickard-Cambridge, 1871 which is morphologically similar to Episinus, is unrelated to it, but instead related to the genus Stemmops O. Pickard-Cambridge, 1894. The genus Thwaitesia O. Pickard-Cambridge, 1881 was recovered as sister to Epsisnus, with strong support (Bayesian PP 97.4%, ML bootstrap 95%). This is similar to the morphological phylogeny of Durán-Barron et  Rodrigues et al. (2022), which places Thwaitesia far from Episinus and Moneta. Unlike our study, which contains both molecular and morphological data, the relationship between the genera was discussed more from an evolutionary perspective. Figure 6. Bayesian analysis of molecular and morphological data of the focal dataset, a matrix excluding all taxa with over 70% missing data. Numbers on nodes are posterior probabilities; bootstrap support from ML analyses is indicated: solid stars indicate bootstrap support values >95%, the gray stars > 50-95%, and nodes with less than 50% support lack stars.

Discussion
Based on the combination of molecular and morphological data, our knowledge of the phylogeny of Theridiidae is rapidly growing (Agnarsson 2004;Liu et al. 2016). The phylogeny serves as a useful tool for comparative studies and as a guide to improved classification. Our phylogeny (Fig. 6) refined that of Liu et al. (2016) by focusing on the genus Episinus. Our results offer the strongest test of the monophyly of Episinus to date and indicate that the genus can be clearly divided into two groups, supported independently by molecular and morphological evidence.
Our results also have implications for the broader phylogeny of Theridiidae in as much as they differ from Liu et al. (2016). As found previously, the placement of the "orphan" pholcommatines Styposis and Phoroncidia is unstable across different analyses, with the current results indicating Styposis is close to Latrodectinae and Phoroncidia is close to Argyrodinae. Latrodectinae includes some of the largest species and of which the genus Steatoda Sundevall, 1833 is polyphyletic. Theridion sangzhiense Zhu, 1998 is here again placed within Pholcommatinae, most likely indicating an error in species identification of GenBank materials. Different from previous studies, we recovered Argyrodinae (plus Phoroncidia) as a sister to Spintharinae plus Hadrotarsinae. If this group is true, these taxa may be united by extreme reduction or loss of webs. Importantly, we recovered Audifia as a member of Hadrotarsinae, as expected based on morphology, but in contrast to Liu et al. (2016). This result indicates the monophyly of Hadrotarsinae, although this subfamily has strong morphological diversity (including two pairs of spermathecae and modifications of spinnerets and palpal claws). The undisputed sister relationship of Anelosiminae and Theridiinae was again supported.
Our study focuses mainly on the subfamily Spintharinae and confirmed its monophyly. The genera Spintharus, Thwaitesia, and Episinus all tend to build an H-shaped web with the animal facing downwards and holding onto parts of the web with its feet, presumably specializing on pedestrian prey such as ants (Agnarsson 2004;Eberhard et al. 2008). A close relationship between Thwaitesia and Epsisnus was proposed by Durán- Barron et al. (2013) based on morphology. However, also based on morphology, Rodrigues et al. (2022), found that Thwaitesia is unrelated to Episinus, and morphological evidence showed that humps are present on the abdomen in Episinus but absent in Thwaitesia. Further evidence for the relationship between Thwaitesia and Episinus may come from the structure of the egg sac (Eberhard et al. 2008). Most theridiid egg sacs have a densely spun outermost layer, while the outermost fibers are loosely woven in Episinus and Thwaitesia (Agnarsson 2004). However, there are insufficient data available on egg sacs of other spintharines.
We found support for the monophyly of Episinus and its division into two morphologically well-defined branches. The "angulatus" group includes E. angulatus (Blackwall, 1836), E. papilionaceous sp. nov., and E. affinis Bösenberg & Strand, 1906. This group differs from the "nubilus" group, which includes E. nubilus Yaginuma, 1960 and E. ornithorrhynchus sp. nov., in the origin of the male embolus and the ventral view of female epigynal. The "angulatus" group can be characterized by the male embolus originating closer to the cymbium at the lateral edge of the palp, whereas in the "nubilus" group the embolus arises in clockwise direction upward bending in the middle of the palp. The "angulatus" group presents only one large atrium in the ventral view of the female epigynal, but the "nubilus" group has two atria separated by the medium septum. The medium septum is a projection in the middle of the atrium and has a guiding function (Yin et al. 2012). In future studies of Episinus and Spintharinae, it will be critical to include other spintharine genera, especially those previously included in Episinus, such as Janula, Neopisinus, and Brunepisinus.