﻿Phylogenetic placement of eight poorly known spiders of Microdipoena (Araneae, Mysmenidae), with descriptions of five new species

﻿Abstract Ten species of the spider genus Microdipoena Banks, 1895 are reported from China, Laos, Indonesia, Georgia, and Seychelles. DNA sequences of the eight species are obtained to confirm their correct identification. The molecular phylogenetic analysis based on five gene fragments (16S, 18S, 28S, COI, and H3) were used to test the relationships and taxonomic placements of eight Microdipoena species, of which five species are documented as new to science: i.e., M.huisunsp. nov. (♀, China), M.lisusp. nov. (♀, China), M.shenyangsp. nov. (♂♀, China), M.thatitousp. nov. (♀, Laos), and M.zhulinsp. nov. (♂♀, China). Five known species are redescribed: M.elsae Saaristo, 1978 (♂♀, Seychelles), M.gongi (Yin, Peng & Bao, 2004) (♂♀, China), M.menglunensis (Lin & Li, 2008) (♂♀, China), M.jobi (Kraus, 1967) (♂♀, Georgia), and M.yinae (Lin & Li, 2013) (♂♀, China). All but M.menglunensis are diagnosed and illustrated. The family Mysmenidae is also the first recorded from Laos and Georgia.


Introduction
Since its inception, the genus Microdipoena Banks, 1895 had a very confusing taxonomic history lasting more than a century. Originally considered as a monotypic genus, it was placed in Theridiidae Sundevall, 1833. Its generotype, Microdipoena guttata Banks, 1895, was designated from Long Island in New York (Banks 1895). Bishop and Crosby (1926) synonymized Microdipoena with Mysmena Simon, 1894 that was accepted by Levi (1956) and Gruia (1977). At almost the same time, Mysmena was transferred from Theridiidae to Symphytognathidae Hickman, 1931 by Forster (1959), and subsequently to Mysmenidae Petrunkevitch, 1928 by Forster andPlatnick (1977). Saaristo (1978) considered that the male palp and epigyne of Microdipoena guttata are quite different in structure and revalidated this genus that consists of two species: M. guttata and M. elsae. Shortly thereafter, Brignoli (1980) reported some Oriental and Australian mysmenids and transferred Mysmena jobi in Symphytognathidae (Kraus 1967), Mysmena illectrix and Mysmena saltuensis in Theridiidae (Simon, 1895) to his created genus Mysmenella Brignoli, 1980. Over the next three decades, some Microdipoena species were placed in so-called "Mysmenella" by other arachnologists (e.g., Baert 1984aBaert , b, 1989Ono 2007;Yin et al. 2004;Li 2008, 2013). Based on the phylogenetic hypothesis of Mysmenidae, Lopardo and Hormiga (2015) re-diagnosed and circumscribed Microdipoena and proposed it synonymized with Anjouanella Baert, 1986 andMysmenella Brignoli, 1980. At this point, the placement and circumscription of this genus has been reasonably confirmed.
Microdipoena is distributed almost worldwide except Antarctica, although it currently consists of only 16 valid species, accounting for about 8.7 percent of 183 mysmenid species (WSC 2023). Known congeners are mainly distributed in Eurasia, Africa, Americas, some Oceanic and Pacific islands.
This paper reports our findings on the study of an inventory specimens collected from China, Laos, Indonesia, Georgia, and Seychelles during 2006 through to 2018, which revealed a total of nine Microdipoena species, including five new to science and four previously known species. The purpose of this study are to sequence five genes of these species, to test their phylogenetic positions and relationships within the genus Microdipoena, and to describe and illustrate the five new Microdipoena species from China and Laos. This paper is also the first report of Mysmenidae from Laos and Georgia.

Species sampling and preservation
Specimens were collected by hand or sifting from leaf litter. All of the specimens were preserved in a 95% ethanol solution at -20 °C. All examined materials and molecular vouchers involved in this study are stored in the Natural History Museum of Sichuan University in Chengdu (NHMSU), China.

Molecular data
To test taxonomic position of these novel species in this study within the Mysmenidae, twelve individuals from eight species were picked out from the examined materials for molecular sequencing. Their prosoma and legs were used to extract genomic DNA and sequence five gene fragments: 16S, 18S, 28S, COI, and H3. Primer pairs and PCR protocols are given in Table 1. The abdomens and male palps were kept as vouchers. Whole genomic DNA was extracted from tissue samples with the TIANamp MicroDNA Kit (TIANGEN) following the manufacturer's protocol for animal tissue. The five gene fragments were amplified in 25 μL reactions. Raw sequences were edited and assembled using BioEdit v. 7.2.5 (Hall 1999). Newly obtained DNA sequence data has been uploaded to GenBank for preservation (accession numbers given in Table 2).
We used these new sequences and a selection from previously sequenced taxa to assemble a partial phylogeny of mysmenid spiders, which only involved five representative genus. A total of 26 mysmenid species was used for phylogenetic analysis ( Table 2). The ingroup includes 11 known, nine undescribed, and four new mysmenid species. Two Maymena species were used as outgroups (see gray region in Table 2). We used the MAFFT v. 7.450 online server (https://mafft. cbrc.jp/alignment/server/) with default parameters to align the sequences of the   eight Microdipoena species. All sequences were concatenated in sequences Matrix v. 1.7.8 (Vaidya et al. 2011). We used PartitionFinder2 (Lanfear et al. 2017) to identify the best-fit models of molecular evolution for each locus. GTR+I+G was selected for COI, H3, 18S, and 28S, and GTR+G was selected for 16S. Topology The maximum likelihood (ML) tree was constructed using Phylosuite v. 1.2.2 (Zhang et al. 2020) with TBR (Tree-Bisection-Reconnection) branch swapping and 2000 bootstrap replicates with default parameters. Bayesian phylogenetic inference (BI) was performed using MrBayes v. 3.2.7 (Ronquist et al. 2012) through the Cipres Science Gateway (Miller et al. 2010) 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 less than 0.01. The Markov chains were sampled every 1000 generations, and the first 25% of sampled trees were burn-in. The program Tracer v. 1.7.1 (Rambaut et al. 2018) was used to analyze the performance of our BI analyses.

Morphological data
Specimens were examined and measured using a Leica M205 C stereomicroscope. Further details were studied with an Olympus BX 43 compound microscope. Male palps and epigynes dissected from the bodies were photographed with a Canon EOS 60D wide zoom digital camera (8.5 megapixels) mounted on an Olympus BX 43 compound microscope. The individual spider was photographed directly under the compound microscope after being reshaped to its natural status. To show more detailed features, epigynes and each disassembled parts of male palps were treated with lactic acid before being embedded in Hoyer`s gum to take photos of the vulvae. The images were montaged using Helicon Focus 3.10.3 (Khmelik et al. 2006) image stacking software.
All measurements are in millimeters. Leg measurements are given as follows: total length (femur, patella, tibia, metatarsus, and tarsus). References to figures in the cited papers are in lowercase (fig. or figs), figures in this paper are noted with an initial capital (Fig. or Figs). Nomenclature of the genital structures was mainly based on Lopardo et al. (2011)

Phylogenetic analysis
The topology inferred by the two different phylogenetic analyses based on the combined sequence dataset of five gene fragments performed (Figs 1,2) show high consistencies in several mysmenid groupings. High support values are common at each end clade. Except for two Maymena species designated as outgroup, the remaining 24 mysmenid species are divided into four major clades, each of which represents a different genus. Both ML and BI trees analyses recovered Microdipoena as monophyletic and a sister group of Mysmena + Trogloneta + Yamaneta.
In the ML tree, the clade of Yamaneta represented by two known species (Y. kehen and K. paquini) is near the base of ML and BI trees, with high support (indicated by yellow box in Fig. 1). The clade of Trogloneta containing three known species (T. granulum, T. yuensis, and T. yunnanensis) is also monophyletic with high support (indicated by pink box in Fig. 1). Four undescribed Mysmena (MYSM-011-ARG, MYSM-018-MAD, and MYSM-028-MAD) and Mysmenidae species (Mysmenidae sp._MD2476) composed the clade of Mysmena shown in the blue box in Fig. 1, which is located in the middle of the topological structure trees, between the clades of Trogloneta and of Microdipoena, and also has relatively high support. A clade composed of ten Microdipoena species (including eight species involved in this study, indicated by red font in the pale box in Fig. 1) and five undescribed species (Fuzhou-Dahuxiang-32, MYSM-006-MAD, MYSM-030-MAD, Mysmeninae sp._7502_050, and AtoL_ARA-GH000003) were monophyletic, but with low support. These results support our taxonomic classification.
The result of BI is consistent with ML for all major clades (Fig. 2). In the BI topology, seven Chinese and one Seychelles species involved in our study (indicated by red font in Fig. 2), together with two known species (Microdipoena nyungwe, M. guttata), two undescribed species (MYSM-030-MAD, AtoL_ARAGH000003), and three undescribed Mysmeninae species (Fuzhou-Dahuxiang-32, MYSM-006-MAD, and Mysmeninae sp._7502_050) form a separate monophyletic, lower supported clade compared to Mysmena, Trogloneta, and Yamaneta. However, each species has high support at each end clade of the BI tree respectively. The available molecular evidence seems sufficient to justify the taxonomic placement of four new and four known Microdipoena species in this study.  Bishop and Crosby 1926: 127). Mysmena Bishop & Crosby, 1926: 177. Microdipoena Saaristo, 1978: 124. Mysmenella Brignoli, 1980: 731 (synonymized by Lopardo and Hormiga 2015. Anjouanella Baert, 1986 Fig. 1. Note the high support of eight species (red font) in the clade of Microdipoena (pale box), which is monophyletic but with low support. Other three clades, Mysmena (blue box), Trogloneta (pink box), and Yamaneta (yellow box), are also monophyletic respectively and with high support. Type species. Microdipoena guttata Banks, 1895 by original designation; type locality Long Island, New York, USA.

Microdipoena elsae Saaristo, 1978
Palp : large, ca as big as ½ size of the carapace. Cymbium translucent, distal end specialized as a broad, collared cymbial conductor, and a small cymbial process, modified by weakly sclerotized folds and a row of stiff short setae (Figs 4D, 5C, 6F-H). Paracymbium smooth, with long setae at the edge. Conductor wide, sclerotized, with two upper and a lower processes (Fig. 6A, B). Tegulum sclerotized, with two upper (a wide, a narrow) and one lower (a narrow) processes (Fig. 6D, E). Embolus filiform, with a membranous hook at the constriction near the middle (Figs 5D, 6C), its distal part coiled into 2.5 loops around cymbial conductor (Figs 4, 5C). Spermatic ducts faintly visible through the surface of palpal bulb and cymbium. Somatic characters (Fig. 3D-F). Coloration: carapace dark brown centrally, yellow-brown marginally. Ocular base black. Chelicera, endites, and labium yellow. Sternum yellow with two brown stripes. Legs yellow and black. Abdomen dark brown with white spots dorsally, yellow with brown spots ventrally. Prosoma: carapace nearly pear-shaped in dorsal view. Cephalic part slightly elevated. Sternum triangular, covered with sparse short setae. Legs: covered with setae and bristles. Femurs I and II with femoral spot. Abdomen: nearly globose.
Epigyne (Fig. 7A-C): spermathecae heavily sclerotized, nearly vertically ovoid, spaced by ca 3× their width. Copulatory duct almost all membranous cystic structure with irregular folds, surround the entirely spermathecae, which enters the spermathecae from posteromedially after gradually harden at the posterior area of spermathecae. Weakly sclerotized fertilization duct starts at the posterolateral side of spermatheca, and then folds back toward the center of vulva (Fig. 7C).
Palp (Fig. 9A-C): The palp 45° inclined to the surface of the tibia. Cymbium translucent, originating prolaterally, with a large cymbial conductor. Paracymbium large, finger-like, with long setae. Tegulum translucent, surface swollen. Embolus thin and relatively short, coiled into one loop over the cymbium, tip with complex structure. Spermatic ducts can be seen through translucent tegulum.
Palp (Fig. 12A-G): The palp 45° inclined to the surface of the tibia. Cymbium translucent, cymbial tooth on the prolateral, sclerotized; the tip specialized as cymbial conductor; a large cymbial process at the contralateral of the cymbial conductor. Paracymbium small, with long setae. Conductor 7-shaped, sclerotized, with two large apophyses apically and two pointed apophyses basally. Tegulum translucent, swollen surface. Embolus wide and long, the tip with complex structure, most structure coiled into two loops. Spermatic ducts can be seen through translucent tegulum.
Somatic characters (Fig. 11D-F). Coloration: carapace dark brown. Ocular base black. Chelicera, endites, labium yellow; sternum yellow with two longitudinal brown stripes. Legs brown-black. Abdomen silvery brown with multiple white spots dorsally, brown with two symmetrical white stripes and multiple little white spots ventrally. Prosoma: carapace nearly pear-shaped in dorsal view. Cephalic part unelevated. Sternum scutiform, slightly plump, covered with sparse setae. Legs: covered with setae and bristles. Femurs I and II with sclerotized femoral spot. Abdomen: nearly spherical in dorsal view, covered with sparse setae.
Distribution. France, Georgia (Adjara), Caucasus, Iran, China, Korea, and Japan.     Etymology. The specific name is derived from the type locality; noun in apposition.
Diagnosis. Male of this new species differs from other congeners by the embolic end twisted into a complex structure, having a cymbial tooth, which is located on the dorsal edge of cymbium, the peculiar shape conductor with a small membranous process and a large, thickened process on lower sides (cf. Figs 12B, C, F, 16A, B, F, 20C, 22B, D, and Lopardo and Hormiga 2015: fig. 132A-F). Female seems similar to M. jobi and M. lisu sp. nov. in the configuration of vulva and having a long scape with wide at basally and weakly sclerotized at distally, but can be distinguished by the smaller spermathecae spaced by ca 3.5× their diameter and the more visible fertilization ducts (cf. Figs 17C, 13C, 14F).
Somatic characters (Fig. 15A-C). Coloration: carapace dark brown. Chelicera, endites, labium dark yellow, sternum yellow with two small brown spots. Legs yellow-brown. Abdomen black with symmetrical white spots dorsally and ventrally. Prosoma: carapace nearly round in dorsal view and peak-shaped in lateral view. Cephalic part elevated and flat. Sternum scutiform, plump, covered with sparse setae. Legs: covered with setae. Mating clasper on metatarsus I, two strong spines on tibia I. Abdomen: nearly globose in dorsal view, cover pale setae.
Somatic characters (Fig. 15D-F). Coloration: carapace pale brown. Ocular base black. Chelicera, endites, labium yellow; sternum yellow with two brown stripes. Legs yellow-brown. Abdomen silvery brown with multiple symmetrical yellow spots dorsally, black with multiple arched yellow stripes and spots ventrally. Prosoma: carapace nearly pear-shaped in dorsal view. Cephalic part slightly elevated. Sternum scutiform, slightly plump, covered in sparse setae. Legs: covered with setae and bristles. Femurs I and II with sclerotized femoral spot. Abdomen: nearly globose in dorsal view, covered with pale setae.
Epigyne ( Etymology. The specific name is derived from the type locality; noun in apposition. Diagnosis. This new species can be distinguished from other congeners by the thickened, long S-shaped fertilization ducts and the entire membranous scape including distal end ( Fig. 18D-F).
Somatic characters (Fig. 18A-C). Coloration: carapace yellow centrally, yellow-brown marginally. Ocular base black. Chelicera, endites yellow, labium yellow, sternum pale yellow with two symmetrical black stripes. Legs yellow-brown. Abdomen silvery yellow dorsally, black with symmetrical white and yellow spots, silvery brown with two symmetrical silvery yellow stripes ventrally. Prosoma: carapace nearly pear-shaped in dorsal view. Cephalic part unelevated. Sternum scutiform, covered with sparse setae. Legs: covered with setae and bristles. Femurs I and II with sclerotized femoral spot. Abdomen: nearly spherical, covered with sparse pale setae. Epigyne (Fig. 18D-F Etymology. The specific name is derived from the Chinese pinyin for bamboo forest (zhú lín), refers to this species living in this habitats; noun in apposition.
Somatic characters (Fig. 21D-F). Coloration: carapace pale yellow centrally, brown marginally. Ocular base black. Chelicera, endites, labium, and sternum yellow. Legs yellow-brown. Abdomen nearly white dorsally, black with multiple white and yellow spots ventrally. Prosoma: carapace nearly pear-shaped in dorsal view. Cephalic part slightly elevated. Sternum scutiform, slightly plump, covered in sparse setae. Legs: covered with setae and bristles. Femurs I and II with sclerotized femoral spot. Abdomen: nearly globose in dorsal view, covered with black setae.
Epigyne (Fig. 23A-D): scape long, with wide folds, tip sclerotized. Copulatory duct membranous, coiled under the spermathecae. Fertilization ducts slightly sclerotized, originating from the ventral side of the epigyne and bent anteriorly. Paired spermathecae nearly round, separated by nearly double their diameter.

Discussion
In this paper, we describe a group of species of the genus Microdipoena that are mainly native to Eurasia. The morphological characteristics of copulatory organs were compared between multiple congeneric species. Some of the diagnostic features they shared were verified, and can be distinguished from those of other genera (cf. male with two or three tibial spines on the leg I; male palp with a paracymbium, distal part of the embolus coiled and distorted into a complex structure in most species; Lopardo and Hormiga 2015). To test whether our taxonomic decisions and their classification status are correct, we also conducted phylogenetic analyses based on molecular evidence for ten named and five undescribed Microdipoena species. Our phylogenetic analysis shows that the monophyly of this genus is valid and these taxonomic judgments proposed by us in this study are correct. However, the male characters of copulatory organs of three species are unknown due to inadequate sampling (M. huisun sp. nov., M. lisu sp. nov., and M. thatitou sp. nov.). According to the reported distribution records, the genus is mainly distributed in the continents of Asia and Africa and nearby islands. Most species of the genus are endemic, some of which have multiple distribution sites (M. elsae, M. nyungwe, M. samoensis), and a few may have expanded distribution ranges as a result of introduction (M. guttata and M. jobi). The origin and diffusion history of Microdipoena are questions worthy of further discussion. Faunal surveys and diversity studies of this genus are a prerequisite for answering these questions, but much work remains to be done. on this manuscript. Ms. Danni Sherwood (Natural History Museum, London, England) kindly checked the English of the manuscript. Thanks to Dr. Shuqiang Li, Dr. Peter W. Jäger, Dr. S. Bayer, Dr. Guo Zheng, Dr. Huifeng Zhao, Dr. Yunchun Li, Dr. Guchun Zhou, and Ms. Xue Sun for their efforts in field sampling.