A Review of the Harvestman Genus Metadentobunus (Opiliones: Sclerosomatidae: Gagrellinae) with A Description of A New Species From Taiwan

Abstract

Ssu-Li Chen and Hsi-Te Shih (2018) The harvestman genus Metadentobunus is endemic to Taiwan, and has two taxa, M. formosae formosae Roewer, 1915 and M. formosae garampiensis Suzuki, 1944. The two subspecies have been raised as two species, and this study redescribes the type species M. formosae based on the type specimen and a neotype of M. garampiensis Suzuki, 1944 is assigned. A new species, Metadentobunus brevispinus sp. nov., is also established from central Taiwan based on morphological and molecular evidence. This new species differs from other congeneric species by having one pair of shorter (than other congeners) spines at frontal margin of the ocularium. It can also be distinguished from M. garampiensis by the basal shape of the penile shaft (constricted vs. parallel), location of the seminal receptacle in ovipositor (5th-6th segment vs. 6th-7th segment) and number of ampullas in the terminal of the seminal receptacle (2 vs. 3). Molecular evidence from mitochondrial 16S rDNA and cytochrome oxidase subunit I (COI) further supports the identity of this new species. An identification key for this genus is included as well.

BACKGROUND

The genus Metadentobunus (Eupnoi: Sclerosomatidae: Gagrellinae) was erected by Roewer (1915) for Metadentobunus formosae Roewer, 1915 (type locality Rokko (= Lugang, Changhua), central Taiwan), as a monotypic genus. This genus is distinguished from other genera of the Gagrellinae from Taiwan by having one pair of frontal spines of the ocularium. Later, Metadentobunus garampiensis Suzuki, 1944 was published from southern Taiwan. After comparing the type specimen of M. formosae, Suzuki (1977) considered M. garampiensis Suzuki, 1944 as a subspecies of M. formosae because the difference between the two taxa is minor. In conclusion, the pedipalpal patella of M. garampiensis has a pronounced apophysis at the inner tip (Suzuki 1944 1977), which is absent in M. formosae according to the original description by Roewer (1915); the frontal spines of ocularium in M. garampiensis is smooth at the tip, which is finely granulated at the tip in M. formosae (Suzuki 1977).

We examined the specimens of Metadentobunus collected extensively from southern and central Taiwan, and found that all have an apophysis at inner tip of each pedipalpal patella. The holotype of M. formosae was also examined, but the pedipalps of the holotype was unfortunately broken from patella to tarsus. We therefore followed the description of Roewer for M. formosae and considered specimens collected from central and southern Taiwan with a pronounced apophysis at the inner tip of the pedipalpal patella are different species. We treat subspecies as species for the taxa mentioned in this study (see Oláh et al. 2018).

Suzuki (1977) pointed out that one character described by Roewer (1915) was incorrect, i.e. there are some tubercles above the first cheliceral segment of M. formosae, but the original description from Roewer is smooth. After we examined the type of M. formosae, we also found that the sex of the holotype is actually female, not male like Roewer (1915) mentioned. As a result, it is necessary to redescribe M. formosae based on the holotype. In addition, because Suzuki (1977: 137) mentioned that the holotype of M. garampiensis was lost, a neotype collected from Sheding, near the type locality Eluanbi, Kenting National Park, was assigned. We also include the description and photos of M. garampiensis females.

After comparing the morphological characters of a series of Metadentobunus specimens collected from central and southern Taiwan, we confirmed that the species from central Taiwan, with different characters, is a new species and describe it in this study using further genetic evidence from the mitochondrial 16S rDNA and COI markers. A key for the genus Metadentobunus is also provided.

MATERIALS AND METHODS

Specimen collection

Specimens collected from central and southern Taiwan (Table 1; Fig. 7) were preserved in 70-95% ethanol, and examined and drawn under a stereomicroscope equipped with a drawing tube. Ovipositors were cleared in hot lactic acid prior to observation of seminal receptacles (Schwendinger and Martens 2002).

Table 1. Haplotypes of 16S rRNA and COI genes of Metadentobunus garampiensis Suzuki, 1944, M. brevispinus sp. nov. from Taiwan. The numbers behind localities are the collecting localities in figure 7.

Species	Locality	NCHUZOOL Catalog no.	Sample size	Haplotype of 16S	DDBJ Access. no. of 16S	Haplotype of COI	DDBJ Access. no. of
M. garampiensis	Kenting National Park, Hengchun, Pingtung [9]	14581	1	Mfg-1	LC433979	Mfg-C1	LC433996
		14582	1	Mfg-2	LC433980	Mfg-C2	LC433997
		14583	1	Mfg-1	LC433979	Mfg-C3	LC433998
	Jhihben Forest Road, Beinan, Taitung [10]	14593	1	Mfg-4	LC433981	Mfg-C4	LC433999
	Tengjhih, Taoyuan, Kaohsiung [11]	14598	1	Mfg-5	LC433982	Mfg-C5	LC434000
M. brevispinus	Sun Moon Lake, Yuchih, Nantou [7]	14585	1	Mdb-1	LC433983	Mdb -C1	LC434001
	Sitou, Lugu, Nantou [6]	14586	1	Mdb-2	LC433984	Mdb -C2	LC434002
		14587	1	Mdb-3	LC433985	Mdb -C3	LC434003
	Huisun Forest Area, Ren-ai, Nantou [8]	14588	1	Mdb-4	LC433986	Mdb -C4	LC434004
	Furong Waterfall, Guosing, Nantou [2]	14589	1	Mdb-5	LC433987	Mdb -C5	LC434005
	Aowanda National Forest Recreation Area, Ren-ai, Nantou [4]	14590	1	Mdb-6	LC433988	Mdb -C6	LC434006
	Dasyueshan Forest Recreation Area, Heping, Taichung [1]	14591	1	Mdb-7	LC433989	Mdb -C7	LC434007
	Tunyuan, Ren-ai, Nantou [3]	14594	1	Mdb-8	LC433990	Mdb -C8	LC434008
	Tongfu, Sinyi, Nantou [5]	14596	1	Mdb-9	LC433991	Mdb -C9	LC434009
outgroups
Gagrella sp. 1	Dasyueshan Forest Recreation Area, Heping, Taichung	14493	1	Gag-1	LC433992	Gag-C1	LC434010
		14494	1	Gag-1	LC433992	Gag-C1	LC434010
Psathyropus sp. 1	Sitou, Lugu, Nantou	14495	1	Prf-1	LC433993	Prf-C1	LC434011
Psathyropusformosa	Lianhuachih, Yuchih, Nantou	14496	1	Prf-2	LC433994	Prf-C2	LC434012
		14497	1	Prf-3	LC433995	Prf-C3	LC434013

The morphological terminology follows Hillyard and Sankey (1990). All measurements are given in mm. Specimens have been deposited in the Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL) and National Museum of Natural Science, Taichung, Taiwan (NMNS). The type specimen of Metadentobunus formosae (SMF 9801162-RI/1162-66) deposited in the Senckenberg Museum in Frankfurt am Main, Germany (SMF) was also examined.

Molecular analyses

Genomic DNA was obtained from appendages (legs, pedipalps, and/or chelicera) using the Epicentre QuickExtract DNA extraction solution 1.0. Polymerase chain reaction (PCR) was used to amplify mitochondrial 16S rRNA and cytochrome oxidase subunits I (COI) genes. Primers of 16H10, 16L29 (Schubart 2009), 1471, 1472 (Crandall and Fitzpatrick 1996), 16Sar, 16Sbr (Palumbi et al. 1991) were used for 16S; and LCO1490, HCO2198 (Folmer et al. 1994), HCOoutout (Prendini et al. 2005) and HCOinner (Chen and Shih 2017) were used for COI. The PCR conditions for the above primers were 35 or 39 cycles of denaturation for 50 s at 94°C, annealing for 70 s at 47°C or 48°C, and extension for 60 s at 72°C, followed by extension for 10 min at 72°C. PCR products were sequenced with Applied Biosystems 3730xl DNA Analyzer, using BigDye Terminator Cycle Sequencing Kits. Sequences were aligned by MUSCLE (Edgar 2004) under default settings, with subsequent manual adjustments. Based on a preliminary phylogenetic analysis, the genera Psathyropus and Gagrella are closely related to Metadentobunus (unpublished data), so we selected Psathyropus formosa (Roewer, 1911), Psathyropus sp. 1 and Gagrella sp. 1 as outgroups (Table 1). Sequences of different haplotypes have been deposited in the DNA Data Bank of Japan (DDBJ) (accession numbers in Table 1).

Phylogenetic congruence between the two dataset partitions was tested under the maximum parsimony criterion using the incongruence length difference (ILD) test (Farris et al. 1994) implemented in the PAUP* program (vers. 4.0b10, Swofford 2003) for a combined analysis of 16S and COI. The parameters included 1000 reiterations of a heuristic search with 100 randomly added sequence replications, TBR branch- swapping, using Steepest Descent and enabling the MULTREES option. The topologies of the two datasets were congruent (P = 0.12) and as such, the sequences were combined.

For the combined dataset, the best-fitting models for sequence evolution of individual datasets were determined by jModelTest (vers. 2.1.4, Guindon and Gascuel 2003; Darriba et al. 2012), selected by the Bayesian information criterion (BIC). The best models obtained for 16S and COI datasets were HKY + G and HKY + I + G, respectively. The Bayesian inference (BI) analysis was performed with MrBayes (vers. 3.2, Ronquist et al. 2012). The search was run with 4 chains for 10 million generations and four independent runs, with trees sampled every 1000 generations. The convergence of chains was determined by the average standard deviation of split frequency values below the recommended 0.01 (Ronquist et al. 2005) and the first 1000 trees were discarded as the burnin accordingly. The maximum likelihood (ML) analysis was conducted in RAxML (vers. 8.0.x, Stamatakis 2006) for the combined dataset. The model GTR + G (i.e. GTRGAMMA) was used for all subsets with 100 runs, and we found the best ML tree by comparing likelihood scores. The robustness of the ML tree was evaluated by 1000 bootstrap pseudoreplicates under the model GTRGAMMA. The pairwise estimates of Kimura two-parameter (K2P) model distance for interspecific and intraspecific genetic diversities were calculated using MEGA (vers. 6, Tamura et al. 2013).

RESULTS

TAXONOMY Family Sclerosomatidae Simon, 1879 Subfamily Gagrellinae Thorell, 1889 Genus Metadentobunus Roewer, 1915 Metadentobunus Roewer, 1915: 149; Roewer 1923: 949; Suzuki 1944: 253; Roewer 1955: 85; Suzuki 1977: 137; Crawford 1992: 30; Li and Song 1993: 241. Type species: Metadentobunus formosae Roewer, 1915, by monotypy. Diagnosis: Abdominal scute with a median spine on second tergite. Pedipalpal tarsus longer than tibia; claw pectinate. Leg II femur with two false articulations. Ocularium with 1 frontal spine on each side. Distribution: Taiwan. Remarks: Metadentobunus is an endemic genus to Taiwan. They are often found on the tree trunk, with camouflage coloration similar with the background.

Key to the genus Metadentobunus

1. Pedipalp with inner apophysis on patella ......................... 2 Pedipalp without inner apophysis on patella ....................... ......................................................................... M. formosae

2. Frontal margin of ocularium with long sharp-pointed spine ................................................................... M. garampiensis Frontal margin of ocularium with short spine ....................... ........................................................ M. brevispinus sp. nov.

Metadentobunus formosae Roewer, 1915

(Fig. 1)

Fig. 1.

Fig. 1. Metadentobunus formosae Roewer, 1915 (holotype, SMF 9801162-RI/1162-66). A, B: dorsal and lateral views of female (body length: 6 mm); C, D: dorsal and lateral views of ocularium; E, F: outer and inner side of distal segment of left chelicera; G: dorsal view of supra-chelicera lamellae; H: ventral view of female genital operculum.

Metadentobunus formosae Roewer, 1915: 149, fig. 81 (type locality: Lugang, Taiwan); Roewer 1923: 949, fig. 1084 (Lugang, Taiwan); Roewer 1955: 85 (Lugang, Taiwan); Suzuki 1970: 33 (list); Wang 1953: 509 (list); Li and Song 1993: 241 (list).

Metadentobunus formosae formosae-Suzuki 1977: 137 (Rokko, Taiwan).

Type material: Holotype: 1 ♀ (SMF 9801162- RI/1162-66), Rokko, Formosa (= Lugang, Changhua, Taiwan).

Diagnosis: Ocularium rectangle, length larger than width, constricted basally, with one pair of sharp and long spines at frontal margin (Fig. 1C- D). Frontal spines of ocularium with tubercles. Patella of pedipalp without inner apophysis at tip (Roewer 1915). Genital operculum protruding from frontal margin (Fig. 1H).

Redescription: Female holotype. Dorsal surface of body granulated. Abdomen with 1 short median spine on the second tergite. Supra- cheliceral lamellae tipped with 4-5 small tubercles (Fig. 1G). Ocularium rectangle (Fig. 1C-D), length larger than width, constricted basally, with one pair of frontal spines, sharp and long. Frontal spines of ocularium finely granulated. All free sternites and genital operculum smooth. Genital operculum protruding from frontal margin (Fig. 1H). Surface of all coxae with scattered coarse granules. Coxa I and IV with a row of humps anteriorly and posteriorly; Coxa II and III with a row of humps anteriorly.

Chelicera. Basal segment with 5-9 tubercles dorsally. Distal segment with setae dorsally, outer side smooth, inner side with setae (Fig. 1E-F).

Pedipalp. Femur with numerous denticles ventrally.

Legs. Trochanters mound-shaped (Fig. 1A-B), femora II with two false articulations, tarsus longer than tibia.

Ovipositor. Unexamined.

Measurements: Holotype female (SMF 9801162-RI/1162-66): Total length of body 6, femur length of leg I-IV 8:15.5:7:12. Length of leg I-IV 33:67:29:45.

Coloration: Not available.

Distribution: Lugang, Changhua, central Taiwan (Fig. 7).

Remark: Metadentobunus formosae is similar to M. garampiensis, except for the morphology of its pedipalpal patella. The inner tip of pedipalpal patella of M. garampiensis has an apophysis at the inner tip of pedipalpal patella, but M. formosae does not.

Metadentobunus garampiensis Suzuki, 1944

(Figs. 2-3)

Fig. 2.

Fig. 2. Metadentobunus garampiensis Suzuki, 1944. Male (neotype, NCHUZOOL 14584). A, B: dorsal and lateral view of neotype; E: supra-chelicera lamellae of male; G: dorsal view of male left pedipalp. Female (NCHUZOOL 14583). C, D: dorsal and lateral view of body; F: supra-chelicera lamellae; H: dorsal view of left pedipalp. Scale bar = 1 mm.

Fig. 3.

Fig. 3. Metadentobunus garampiensis Suzuki, 1944. Male (neotype, NCHUZOOL 14584). A, B: inner and outer side of left pedipalp; D, E: dorsal and lateral views of ocularium; G, H: ventral and lateral views of penis; I: different morphology of penis from Tengjhih, ventral view (NCHUZOOL 14598); J: inner side of left chelicera; K: outer side of left chelicera. Female (NCHUZOOL 14583). C: inner side of female left pedipalp; F: lateral view of ocularium; L: female seminal receptacle (NCHUZOOL 14501); M: ventral view of female genital operculum (NCHUZOOL 14583). s: stylus; g: glans; ap: alate part. Scale bars: A-C, G-K, M = 1 mm; D, E, F = 0.5 mm; L = 0.1 mm.

Metadentobunus garampiensis Suzuki, 1944: 253-255, figs. 4-5 (type locality: Oluanpi, Kenting National Park, Taiwan); Suzuki 1970: 33 (list).

Metadentobunus formosae garampiensis-Suzuki, 1977: 137- 139, fig. 7 (Kenting Botanical Garden, Kenting National Park, Taiwan); Li and Song 1993: 241 (list).

Type material: Neotype: 1 ♂ (NCHUZOOL 14584), Sheding, Kenting National Park, Hengchun, Pingtung, Taiwan, 21°57'30.69"N, 120°48'42.41"E, elevation of 206 m, coll. S.-P. Wu, 16 July 2016.

Other material examined from Taiwan: 1 ♂ (NCHUZOOL 14498) and 1 ♂ (NCHUZOOL 14598), Tengjhih, Taoyuan, Kaohsiung, 23°03'54.74"N, 120°45'34.79"E, elevation of 1616 m, coll. S.-L. Chen, 9 May 2015; 1 ♂ (NCHUZOOL 14499) and 1 ♀ (NCHUZOOL 14597), Tengjhih, Taoyuan, Kaohsiung, 23°03'54.74"N, 120°45'34.79"E, elevation of 1616 m, coll. S.-L. Chen, 7 Sep. 2014; 2 ♀ (NCHUZOOL 14500) and 1 ♀ (NCHUZOOL 14583), Kenting National Park, Hengchun, Pingtung, 21°57'30.69"N, 120°48'42.41"E, elevation of 206 m, coll. S.-P. Wu, 26 July 2016; 1 ♀ (NCHUZOOL 14501), Kenting National Park, Hengchun, Pingtung, 21°57'30.69"N, 120°48'42.41"E, elevation of 206 m, coll. S.-P. Wu, 24 July 2016; 1 ♀ 3 Juveniles (NCHUZOOL 14502), Kenting National Park, Hengchun, Pingtung, 21°57'30.69"N, 120°48'42.41"E, elevation of 206 m, coll. S.-P. Wu, 23 Aug. 2016; 1 ♂ (NCHUZOOL 14581) and 1 ♀ (NCHUZOOL 14582), Kenting National Park, Hengchun, Pingtung, 21°57'30.69"N, 120°48'42.41"E, elevation of 206 m, coll. H.-Y. Chang, 19 Aug. 2015; 1 ♀ (NCHUZOOL 14593), Jhihben Forest Trail, Beinan, Taitung, coll. S.-P. Wu, 29 Apr. 2016.

Etymology: The name garampiensis was derived from the Japanese pronunciation of type locality (Oluanpi = Eluanbi = Ngoluanpi, in Kenting National Park).

Diagnosis: Male: ocularium with one pair of sharp and long spines at frontal margin (Fig. 3D- E). Patella of pedipalp with an inner apophysis at tip (Fig. 2G). Patella of pedipalp with numerous sharp-tipped tubercles dorsally. Shaft long, parallel at base (Fig. 3G). Female: seminal receptacle at 6th-7th segment of ovipositor, with three ampullas terminally (Fig. 3L). Genital operculum of female protruding from frontal margin (Fig. 3M).

Description: Male neotype. Dorsal surface of body granulated. Supra-cheliceral lamellae tipped with 4-5 small tubercles (Fig. 2E). Abdomen with 1 short median spine on the second tergite. Ocularium rectangle, length larger than width, constricted basally, with one pair of sharp and long spines at frontal margin (Fig. 3D-E). Frontal spines of ocularium smooth except sparse short hairs. All free sternites and genital operculum smooth. Surface of all coxae with scattered coarse granules. Coxa I and Coxa IV with a row of humps anteriorly and posteriorly; Coxa II and Coxa III with a row of humps anteriorly.

Chelicera (Fig. 3J-K). Basal segment with a ventral spur below, 14-15 tubercles dorsally and a row of tubercles at inner side. Distal segment with setae above, outer side smooth, inner side with setae.

Pedipalp (Fig. 3A-B). All segments with lots of setae, excepting trochanter. Trochanter with 4 tubercles ventrally and 1 spine-like tubercles dorsally. Femur with numerous tubercles ventrally, inner side with a row of tubercles. Patella thickened distally, with an inner apophysis at tip, numerous sharp-tipped tubercles dorsally (Fig. 2G). Tibia covered with many simple setae especially in inner tip. Tarsus elongated, with a row of tubercles at inner side (Fig. 3A). Claw pectinate.

Legs. All legs slim and elongate. Trochanters mound-shaped (Fig. 2A-B), femora II with two false articulations, tarsus longer than tibia.

Penis (Fig. 3G-H). Shaft 2.6 long, 0.28 wide at base, 0.28 wide at middle; glans 0.23 long, 0.1 wide at base. Shaft long, parallel at base; alate part with sacs (Fig. 3G). Glans widest at base. Stylus acicular.

Female (NCHUZOOL 14583): Dorsal surface of body similar to male, excepting abdomen of female longer than male (Fig. 2C-D). Frontal spines of ocularium with tubercles and sparse short hairs. Supra-cheliceral lamellae tipped with 3-4 small tubercles (Fig. 2F). Trochanter of pedipalps with 4 tubercles ventrally. Pedipalpal femur with a longitudinal row of tubercles at inner side, tibia normal with covering of many hairs entirely (Fig. 3C). Inner-tip apophysis of pedipalpal patella longer than male. Pedipalpal tibia of female with a small apophysis at inner tip (Fig. 2H). Genital operculum protruding from frontal margin (Fig. 3M). Seminal receptacle (NCHUZOOL 14501, Fig. 3L; NCHUZOOL 14593) located in 6th-7th segment of ovipositor, terminal with three ampullas.

Measurements: Neotype male (female of NCHUZOOL 14583): Cephalothorax 1.12 (1.58) long, 2.72 (3.45) wide; abdomen 2.73 (3.87) long, 2.84 (3.68) wide. Total length of body 3.85 (5.45). Measurements of left pedipalp and legs are shown in table 2.

Table 2. Pedipalp (left) and legs (left) measurements (in mm) of the neotype male(NCHUZOOL 14584) of Metadentobunus garampiensis Suzuki, 1944. Measurements of female (NCHUZOOL 14583) are shown in parentheses.

	Trochanter	Femur	Patella	Tibia	Tarsus	Total
Pedipalp	0.33 (0.47)	1.41 (1.17)	0.52 (0.61)	0.77 (0.92)	1.35 (1.44)	4.38 (4.61)
Leg I	0.47 (0.44)	8.32 (6.55)	1.12 (1.15)	7.46 (5.92)	19.28 (16.34)	36.65 (30.4)
Leg II	0.49 (0.49)	17.97 (14.02)	1.66 (1.51)	18.71 (14.66)	49.44 (41.33)	88.27 (72.01)
Leg III	0.51 (0.51)	7.53 (6.24)	1.29 (1.05)	6.79 (5.53)	20.14 (17.24)	36.26 (30.57)
Leg IV	0.54 (0.57)	12.26 (10.73)	1.75 (1.31)	10.58 (8.47)	31.55 (25.79)	56.68 (46.87)

Coloration: Male. Dorsum golden yellow with granules (Fig. 2A-B). Carapace golden yellow with small light brown flecks along frontal and lateral edge. Ocularium golden yellow, with white eye rings. Coxa of all legs golden yellow, edge brown. Genital operculum golden yellow, light yellow base, distal edge light yellow, edge brown. Sternites pale yellow, whiten posterior. Chelicerae pale yellow, basal segment with brown spots dorsally. Pedipalps pale yellow, with femora and patellae brown. All legs brown with golden yellow rings. Female similar with male, but darker (Fig. 2C-D).

Variation: One male specimen (NCHUZOOL 14581) has frontal spines with finely granulated tip on ocularium. In addition, the specimen (NCHUZOOL 14598) from Tengjhih have slightly different morphology of penis, i.e. alate part with longer sacs (Fig. 3I).

Distribution: Southern Taiwan (Fig. 7).

Habitat: The specimens were found on tree trunks, which appeared to be one of their habitats.

Remarks: After examining of a series of specimens, we found the character of frontal spines on ocularium with finely granulated tip is variable, which is different from Suzuki’s description (1977).

Metadentobunus brevispinus sp. nov.

(Figs. 4-5)

Fig. 4.

Fig. 4. Metadentobunus brevispinus sp. nov. A, B: dorsal and lateral views of male (holotype, NCHUZOOL 14599); C, D: dorsal and lateral views of female (paratype, NCHUZOOL 14600); E: color in live, male and female. Scale bar = 1 mm.

Fig. 5.

Fig. 5. Metadentobunus brevispinus sp. nov. Male (holotype, NCHUZOOL 14599). A, B: inner and outer side of left pedipalp; D, E: dorsal and lateral views of ocularium; F, G: ventral and lateral views of penis; J: inner side of left chelicera; K: outer side of left chelicera; L: supra-chelicera lamellae of male; H: different morphology of penis from Dasyueshan Forest Recreation Area, ventral view (NCHUZOOL 14505). Female (paratype, NCHUZOOL 14600). C: inner side of female left pedipalp; I: female seminal receptacle; M: supra-chelicera lamellae of female; N: dorsal view of left pedipalp; O: dorsal view of left pedipalp; P: ventral view of female genital operculum. s: stylus; g: glans; ap: alate part. Scale bars: A-C, J-K, F-H, L-P = 1 mm; D, E = 0.5 mm; I = 0.1 mm.

urn:lsid:zoobank.org:act:0CBDAE16-E48C-4753-A40A- 0E09999E4DEF

Type material: Holotype: 1 ♂ (NCHUZOOL 14599), Sitou, Lugu, Nantou, Taiwan, 23°40'27.92"N, 120°47'45.19"E, elevation of 1107 m, coll. S.-L. Chen, 1 Dec. 2013. Paratypes: 1 ♀ (NCHUZOOL 14600), 1 ♂ (NCHUZOOL 14586) , 1 ♀ (NCHUZOOL 14587), 1 ♂ (NMNS 7926-001) and 1 ♀ (NMNS 7926-002), same data as holotype; 1 ♂ (NCHUZOOL 14585), Sun Moon Lake (= Rihyuetan), Yuchih, Nantou, Taiwan, 23°51'26"N, 120°56'42"E, elevation of 764 m, coll. H.-Y. Chang, 19 Jan. 2014; 1 ♂ (NCHUZOOL 14595) and 1 ♂ (NCHUZOOL 14596), Tongfu, Sinyi, Nantou, Taiwan, 23°31'23"N, 120°53'60"E, elevation of 2250 m, coll. S.-L. Chen, 15 July 2015.

Other material examined from Taiwan: 1 ♀ (NCHUZOOL 14503), Sitou, Lugu, Nantou, 23°40'29.15"N, 120°47'48.55"E, elevation of 1107 m, coll. S.-L. Chen, 1 Dec. 2013; 4 ♂ 3 ♀ (NCHUZOOL 14504), Tongfu, Sinyi, Nantou, 23°31'23"N, 120°53'60"E, elevation of 2250 m, coll. S.-L. Chen, 15 July 2015; 1 ♀ (NCHUZOOL 14588), Huisun Forest Area, Ren-ai, Nantou, 24°05'26.55"N, 121°01'58.48"E, elevation of 688 m, coll. S.-L. Chen, 14 Nov. 2014; 1 ♀ (NCHUZOOL 14589), Furong Waterfall, Guosing, Nantou, 24°05'31.14"N, 120°56'13.95"E, elevation of 636 m, coll. S.-L. Chen, 14 Sep. 2014; 1 ♂ (NCHUZOOL 14590), Aowanda National Forest Recreation Area, Ren-ai, Nantou, 23°56'43.20"N, 121°10'58.01"E, elevation of 1243 m, coll. S.-L. Chen, 1 Oct. 2013; 1 ♂ (NCHUZOOL 14505), Dasyueshan Forest Recreation Area, Heping, Taichung, 24°15'22.32"N, 121°00'24.82"E, elevation of 2240 m, coll. S.-L. Chen, 26 Nov. 2013; 1 ♀ (NCHUZOOL 14591), Dasyueshan Forest Recreation Area, Heping, Taichung, 24°15'22.32"N, 121°00'24.82"E, elevation of 2240 m, coll. S.-L. Chen, 23 Aug. 2014; 1 ♂ (NCHUZOOL 14592), Cingjing, Ren-ai, Nantou, 24°03'26.19"N, 121°09'47.48"E, elevation of 1891 m, coll. S.-L. Chen, 3 Aug. 2014; 1 ♀ (NCHUZOOL 14594), Tunyuan, Ren-ai, Nantou, 24°03'1.16"N, 121°12'54.48"E, elevation of 1590 m, coll. S.-L. Chen, 16 June 2015.

Etymology: The name brevispinus is derived from the Latin brevi and spinus, which refers to the one pair frontal spines on ocularium of this species are shorter (Fig. 5D-E) than the other species of the genus Metadentobunus (Figs. 1C-D; 3D-E).

Diagnosis: Male: ocularium with one pair of short frontal spines (Fig. 5D-E). Patella of pedipalp with inner apophysis at tip (Fig. 5N-O). Shaft long, constricted at base (Fig. 5F). Female: seminal receptacle at 5th-6th segment of ovipositor, with two ampullas terminally (Fig. 5I). Genital operculum flat at frontal margin (Fig. 5P).

Description: Male holotype. Dorsal surface of body granulated. Supra-cheliceral lamellae tipped with 5 small tubercles (Fig. 5L). Abdomen with 1 short median spine on the second tergite. Ocularium rectangle (Fig. 5D), length slightly larger than width, constricted basally, with short spine at frontal margin (Fig. 5E). Frontal spines of ocularium smooth except sparse hairs. All free sternites and genital operculum smooth. Surface of all coxae with scattered coarse granules. Coxa I and IV with a row of humps anteriorly and posteriorly; Coxa II and III with a row of humps anteriorly.

Chelicera (Fig. 5J-K). Basal segment with 7 tubercles and setae above, with a slender spur ventrally. Distal segment with setae above, outer side smooth, inner side with setae.

Pedipalp (Fig. 5A-B). All segments with lots of setae, excepting trochanter. Trochanter with 1 conspicuous tubercle. Femur with conspicuous tubercles ventrally, with 11 tubercles on outer side (Fig. 5B) and a row of tubercles on inner side (Fig. 5A). Patella thickened distally, with an inner apophysis at tip. Tibia covered with many simple setae especially in inner tip. Tarsus elongated, with a row of tubercles at inner side (Fig. 5A). Claw pectinate. Legs. All legs slim and elongate. Trochanters mound-shaped (Fig. 4A-B), femora II with two false articulations, tarsus longer than tibia.

Penis (Fig. 5F-G). Shaft 2.28 long, 0.21 wide at base, 0.29 wide at middle; glans 0.2 long, 0.1 wide at base. Shaft long, constricted at base; alate part with sacs (Fig. 5F). Glans widest at base. Stylus acicular.

Female paratypes (NCHUZOOL 14600, NMNS 7926-002): Dorsal surface of body similar to male, excepting abdomen of female longer than male (Fig. 4C-D). Frontal spines of ocularium smooth except sparse hairs. Supra-cheliceral lamellae tipped with 2-4 small tubercles (Fig. 5M). Trochanter of pedipalps with 1 conspicuous tubercle ventrally. Pedipalpal femur with a row of black-tipped tubercles at inner side, tibia covered with simple setae entirely (Fig. 5C). Inner-tip apophysis of pedipalpal patella longer than male. Pedipalpal tibia of female with small apophysis at inner tip (Fig. 5O). Genital operculum flat at frontal margin (Fig. 5P). Seminal receptacle (Fig. 5I) located in 5th-6th segment of ovipositor, terminal with two ampullas.

Measurements: Holotype male (female paratype, NCHUZOOL 14600): Cephalothorax 1.15 (1.68) long, 2.62 (3.0) wide; abdomen 2.16 (3.78) long, 2.41 (3.39) wide. Total length of body 3.31 (5.46). Measurements of left pedipalp and legs are shown in table 3. Female: similar with male, but darker than male.

Table 3. Pedipalp (left) and legs (left) measurements (in mm) of the holotype male(NCHUZOOL 14599) of Metadentobunus brevispinus sp. nov. Measurements of female (NCHUZOOL 14600) are shown in parentheses.

	Trochanter	Femur	Patella	Tibia	Tarsus	Total
Pedipalp	0.32 (0.37)	1.08 (1.18)	0.53 (0.56)	0.85 (0.8)	1.17 (1.41)	3.95 (4.32)
Leg I	0.4 (0.44)	7.7 (6.13)	0.93 (1.02)	6.34 (5.31)	17.52 (15.75)	32.89 (28.65)
Leg II	0.42 (0.44)	14.22 (13.09)	1.01 (1.05)	13.85 (13.26)	42.42 (39.74)	71.92 (67.58)
Leg III	0.41 (0.46)	5.81 (5.62)	0.92 (1.03)	5.31 (4.46)	17.29 (15.14)	29.74 (26.71)
Leg IV	0.42 (0.5)	9.5 (9.11)	1.28 (1.08)	8.28 (6.87)	27.45 (25.35)	46.93 (42.91)

Coloration: Male. Dorsum golden yellow with granules, spine on second tergite black (Fig. 4A- B). Carapace golden yellow with small light brown flecks along frontal and lateral edge. Ocularium golden yellow, with white eye rings. Coxa of all legs golden yellow. Genital operculum golden yellow, light yellow base, distal edge light yellow. Sternites pale yellow, whiten posterior. Chelicerae pale yellow, basal segment with brown spots. Pedipalps pale yellow, with femora and patellae brown. All legs brown with golden yellow rings. Female similar with male, but darker (Fig. 4C-D).

Variation: One female specimen (NCHUZOOL 14588) has frontal spines with finely granulated tip on ocularium. In addition, the female specimen (NCHUZOOL 14591) from the Dasyueshan Forest Recreation Area has slender and long penile shaft and the different morphology of penile alate part (Fig. 5H).

Distribution: Central Taiwan (Fig. 7).

Habitat: This species can be found on the tree trunks, sometimes on the artificial wooden guideboard.

Remarks: This new species can be separated from other congeneric species by having one pair of shorter (than other congeners) spines at frontal margin of the ocularium (Fig. 5D-E, Q). It can be also distinguished from M. garampiensis by the number of tubercles on the dorsal basal segment of the chelicera (7 vs. 14-15) (Fig. 5J-K vs. Fig. 3J- K), the basal shape of the penile shaft (constricted vs. parallel) (Fig. 5F vs. Fig. 3G), location of the seminal receptacle in ovipositor (5th-6th segment vs. 6th-7th segment) and number of ampullas in the terminal of seminal receptacle (2 vs. 3) (Fig. 5I vs. Fig. 3L).

DNA analyses

A 451 base-pair (bp) segment of 16S and 624 bp segment of COI from 19 specimens of the genus Metadentobunus and outgroups were amplified and aligned (Table 1). No specimen of M. formosae was collected and therefore the species was not be included in the molecular analysis. The phylogenetic tree of the combined genes was constructed by BI (Fig. 6), and the bootstrap support values from ML analysis was also shown on the tree. It is clear that the genus Metadentobunus is monophyletic with high support by both BI and ML methods. There are two clades within Metadentobunus with high BI support, and they are referred to as M. garampiensis and M. brevispinus sp. nov., respectively.

Fig. 6.

Fig. 6. A Bayesian inference (BI) tree for the species of Metadentobunus from Taiwan and outgroups, based on the combined dataset of 16S and COI genes. Probability values at the nodes represent the support values for BI and maximum likelihood (ML). Numbers behind the collection sites correspond to the sites in figure 7.

Fig. 7.

Fig. 7. The collection sites for the species of Metadentobunus in Taiwan. Numbers beside the symbols correspond to the collection sites in table 1 and figure 6. In addition, the numbers 12 and 13 mean the sites for Cingjing, Nantou and Lugang (= Rokko), Changhua, respectively.

With regard to genetic divergence, the maxi- mum intraspecific and the minimum interspecific COI distances of Metadentobunus are 2.63-5.03% and 6.79%, respectively (Table 4).

Table 4. Matrix of percentage pairwise nucleotide divergences with K2P distance based on 16S rRNA and cytochrome c oxidase I (COI) genes of M. garampiensis Suzuki, 1944 and M. brevispinus sp. nov. In the right half, the lower left values are 16S and the upper right ones are COI. Values of range are shown in parentheses.

Species	Intraspecific		Interspecific
	16S	COI	M. garampiensis	M. brevispinus
M. garampiensis	0.64 (0-1.15)	1.49 (0.16-2.63)	-	11.08 (6.79-13.85)
M. brevispinus	1.23 (0-2.54)	2.44 (0.87-5.03)	4.72 (4-5.42)	-

DISCUSSION

Metadentobunus was established by Roewer in 1915; however, Suzuki (1977) and our study found some incorrect descriptions in Roewer (1915), e.g. the morphology of chelicerae and the sex of type specimen. With regard to the type locality of M. formosae, we surveyed Lugang several times, but did not find any specimen of this species. One possibility is that its habitat in Lugang has been destroyed because this area is now a developed town. Another possibility is that the specimen might be collected from other unknown places in Taiwan or other regions, and was actually exported to Lugang, because Lugang was an international harbor in the 1910s.

Based on the series of specimens of M. brevispinus sp. nov., some variation can be observed in the penile alate part among specimens from different areas, although these morphological differences belong to intraspecific variation without further subdivision (Fig. 6). For example, specimens from the Dasyueshan Forest Recreation Area (“Mdb-7 + Mdb-C7” in Fig. 6) and Sitou (“Mdb-2 + Mdb-C2” in Fig. 6) belong to the same clade, but the penile alate part between specimens from the two areas is different (Fig. 5H vs. Fig. 5F). Similarly, specimens of M. garampiensis from Tengjhih (“Mfg-5 + Mfg-C5” in Fig. 6) and those from Kenting (“Mfg-1 + Mfg-C1” in Fig. 6) have different penile alate morphology (Fig. 3I vs. Fig. 3G). Apparently, there are intraspecific variations in the male penile alate part in both M. brevispinus and M. garampiensis. Despite this, M. brevispinus can also be distinguished from M. garampiensis by the basal shape of the penile shaft (constricted vs. parallel) (Fig. 5F, H vs. Fig. 3G, I), location of the seminal receptacle in ovipositor (5th-6th segment vs. 6th-7th segment) and number of ampullas in the terminal of the seminal receptacle (2 vs. 3) (Fig. 5I vs. Fig. 3L).

In terms of COI distance in Metadentobunus species, the minimum interspecific distances is 6.79% (Table 4), which is smaller than that on harvestmen from Germany (7.5%; Astrin et al. 2016) and the Pseudogagrella from Taiwan (9.08%; Chen and Shih 2017). Despite this, the maximum intraspecific distances are 2.63% and 5.03% for M. garampiensis and M. brevispinus, respectively, both of that are smaller than 6.79%. Due to the gap between intraspecific and interspecific COI distances exist, this makes the species delimitation of M. brevispinus clear. In addition, the phylogenetic tree (Fig. 6) has confirmed that both M. garampiensis and M. brevispinus are two clades, with high support. Therefore the mitochondrial 16S and COI (Table 4; Fig. 6) shows that M. brevispinus and M. garampiensis are distinct enough in genetic evidence. Some authors considered the mitochondrial markers to be limited in delimiting recently diverged species, and additional nuclear markers are suggested (Hickerson et al. 2006; Satler et al. 2013; Smith et al. 2018). Further studies using nuclear markers to test if there is reproductive isolation between the two species and extensive surveys for their distributional ranges can clarify the species delimitation in depth.

In conclusion, according to the ocularium, pedipalp and penis characters, we describe anew species from central Taiwan, M. brevispinus, with molecular evidence from the mitochondrial 16S and COI. This increases the number of species in the genus Metadentobunus from Taiwan to three.