Two new species of the genus Macrothele Ausserer, 1871 from Yunnan Province, China (Araneae: Macrothelidae)

. Two new species of the genus Macrothele Ausserer, 1871 are described from Yunnan Province, China: Macrothele nullispine sp. nov. and M. auriculata sp. nov. Detailed descriptions, photos of morphological characteristics, DNA barcode sequences and genetic distances of the new species are provided.


Introduction
The genus Macrothele Ausserer, 1871 of the family Macrothelidae, Simon, 1892 is a genus of mygalomorph spiders with venom, considered medically significant.So far, 49 described species of Macrothelidae have been reported worldwide: 43 species in Macrothele, and 6 species in Vacrothele Tang & Yang, 2022.Macrothele is found in Asia, Europe, and Africa (World Spider Catalog 2023), while Vacrothele Tang & Yang, 2022 is restricted to Asia only (Tang et al. 2022).
Macrothele generally live on slopes or cliffs with good ventilation and unobstructed drainage.The retreats of Macrothele and Vacrothele are quite different, with the former using existing rock crevices along roadsides or cracks in soil slopes for nesting, and the latter making burrows in loose soil.
The two new species of Macrothele proposed here were collected from Yunnan Province, China.The results of morphological classification and DNA sequencing analyses suggest they are not known to science so far, and are herein named Macrothele nullispine sp.nov.and M. auriculata sp.nov.Type specimens are deposited in the Institute of Entomoceutics Research, Dali University, China (DUIER).

Sample processing
All examined specimens were preserved in 80% ethanol.The left male palps were removed from the specimens.After taking habitus photos first, the palpal bulb was then separated from the distal part of the palpal tibia and stored in 80% ethanol; the female spermathecae were first dissected from the opisthosoma using a forceps and a dissecting needle, and then treated in 10% NaOH for 24 hours to dissolve the non-sclerotized surrounding tissue.Specimens were examined and measured with Olympus SZX16 and Leica M205A stereo microscopes and an Olympus CX33 compound microscope.All measurements are in millimeters.Leg measurements are given as following: total length (femur + patella + tibia + metatarsus + tarsus). 1 male, 1 female).Each sample took about 10 mg of spider leg tissue.DNA extraction was conducted using Tiangen blood/cell/tissue/genomic DNA extraction kit.After extraction, PCR amplification of the COI gene was conducted using the universal primers: LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCOoutout (5'-GTAAATATATATGRTGDGCTC-3') (Folmer et al. 1994).PCR reaction system of 50 μL: Gold Mix (green) 45 μL, 2 μL each of forward and reverse primers, 1 μL of DNA template.Amplification procedure: 98°C pre-deformation for 2 min; 98°C for 10 s, 46°C for 10 s, 72°C for 10 s for 30 cycles; 72°C extension for 1 min.The PCR amplification products were detected by 1.0% agarose gel electrophoresis and the results were visualized using a Bio-Rad gel imaging system.The sequencing quality was observed using SeqMan (DNASTAR, Inc. ver.7.1.0.44), the sequences were proofread, spliced, and then subjected to BLAST homology analysis in the NCBI database.Genetic distances between species were calculated using MEGA 7 (Kumar et al. 2016), and the resulted sequences were uploaded to NCBI to obtain GenBank numbers.

Diagnosis
This new species is similar to Macrothele arcuata Tang, Zhao & Yang, 2020 (Tang et al. 2020), but differs by the male embolus and the bulb not strongly bent in the new species, and the embolus gradually tapering down from the bulb, with a curved apex (Fig. 3D-G), and the palpal tibia without spines (Fig. 3A-C); male chelicerae ventrally with 9 short and stout teeth (Fig. 2D); female copulatory ducts long, with a wider base and an inwardly curved proximal end, spermathecae small and nearly dropletlike (Fig. 4D-E).Conversely, the embolus and bulb are strongly bent, the end of the embolus is straight in; and the female copulatory ducts are longer the M. arcuata.

Natural history
The spiders mainly reside in existing stone crevices or caves along highways, soil slopes, gaps in tree bark, and under fallen leaves.

Diagnosis
This new species is similar to Macrothele guihzouensis Hu & Li, 1986(Wu et al. 2022), but differs by the male embolus being slender, slightly curved from the middle with a pointed apex in the new species (Fig. 7D-F); the palp tibia with six prolateral spines and three dorsal spines (Fig. 7A-B), the retrolateral face of the palpal trochanter has no lyrate organ; female copulatory ducts long (Fig. 8D).Conversely, the embolus is short, stout, and straight in M. guihzouensis, and with a flattened apex; the retrolateral face of the palpal trochanter has a lyrate organ; and the female copulatory ducts are short.

Etymology
The new species name is a noun in apposition and refers to the auricle-like female genitalia.7) MeasureMents.Total length 18.10: cephalothorax 6.95 long, 5.59 wide; opisthosoma 8.66 long, 6.02 wide.

Natural history
The spiders mainly reside in existing stone crevices or caves along highways, soil slopes, gaps in tree bark, and under fallen leaves.

Discussion
Table 2 shows that the intraspecific genetic distance between the two samples of M. nullispine sp.nov. is 0.002, which is less than 2%, the interspecific genetic distance between M. nullispine and M. arcuata Tang, Zhao & Yang, 2020 is 0.544, significantly greater than the intraspecific genetic distance, so there is a clear gap between the two samples (Hebert et al. 2004).Therefore, we consider M. nullispine sp.nov.as a new species.
Table 3 shows that the intraspecific genetic distance between the two samples of M. auriculata sp.nov. is 0.001, which is less than 2%, the interspecific genetic distance between M. auriculata and M. guizhouensis Hu & Li, 1986 is 0.186 ~ 0.187, significantly greater than the intraspecific genetic distance, there is a clear gap between the two samples.Therefore, we consider the M. auriculata as a new species.
Morphologically, the two new species described are different from other species.In addition to morphological observations, the interspecific genetic distance was calculated, and the results indicate that both species are indeed new species in the genus Macrothele.

Table 2 .
Intraspecific genetic distances in two samples of Macrothele nullispine sp.nov.based on the p-distance mode.

Table 3 .
Intraspecific genetic distances in two samples of Macrothele auriculata sp.nov.based on the p-distance mode.