Pseudohynobius puxiongensis ( Fei et Ye, 2000 )

Xiong, Jian-Li, Gu, Hai-Jun, Gong, Tian-Jian & Zeng, Xiao-Mao, 2011, Redescription of an enigmatic salamander, Pseudohynobius puxiongensis (Fei et Ye, 2000) (Urodela: Hynobiidae), Zootaxa 2919, pp. 51-59 : 52-58

Pseudohynobius puxiongensis ( Fei et Ye, 2000)

Diagnosis. This species differs from all other species of the genus by the following combination of features: dorsal color gray-yellow with yellowish spots, dorsal tail with distinct yellowish line ( Fig. 1 A); TOL shorter than congeners; TL <SVL, HL / HW <1.53, and moderate number of vomerine teeth (11–15) ( Table 2 ).

Redescription of the species. External morphology. The morphological description is based on the four adult males. Body slender, TOL 108.5–137.5 mm ( Table 1 ). Head moderately depressed, HL>HW. Tongue large and elliptical. Snout rounded in dorsal view, gently sloping from eye to nostrils. Nostril small, near tip of snout. Eye large, distinctly protuberant; DE slightly less than SL. Eyelid present, no labial fold; the gular fold distinct. Angle of jaw just posterior to rear corner of eye with a protuberance from angle of jaw to gular fold. Longitudinal groove present on protuberance from the angle of jaw to the gular fold and intersecting with transverse groove of angle of jaw ( Fig. 1 A). A “V”-shaped protuberance present on the posterior eyelid, and extending posteriorly to the middle of the tip of head.

Traits Ps. puxiongensis Ps. flavomaculatus 1 Ps. kuankuoshuiensis 2 1Data for P. flavomaculatus are from Fei and Ye (1982). 2Data for P. kuankuoshuiensis are from Xu et al. (2007).3 Data for P. shuichengensis are from Tian et al. (1998). 4Data for P. jinfo are from Wei et al. (2009). 5Data for P. guizhouensis are from Li et al. (2010).

Body cylindrical and slender, with 11 costal grooves. The skin of the dorsum and venter slippery, scattered with many microscopic granular glands. Dorsum with distinct groove from the back of head to the base of tail. Feeble longitudinal groove present in the middle of the belly ( Fig. 1 B). Cloaca rounded with a longitudinal groove, abundant yellowish pigment at the posterior cloaca, little pigmented at the anterior cloaca, and central cloaca protruded to form a papillate. Limbs well developed, the hindlimb slightly longer and more robust than forelimb. When forelimb and hindlimb are adpressed to the side of the trunk, tips of digits meet or slightly overlap. In order of decreasing length, fingers 2-3-4-1; toes 3-4-2-5-1. Tips of fingers and toes rounded, without horny cover. No palmar or tarsal tubercles or digital webbing.

Tail short, less than SVL, slightly wide than more than high, cylindrical at base, flattened gradually toward the tip, and strongly tapered in the end. Upper tail fin fold feeble, originating at a point 3/4 of the distance from base to tip of tail, gradually increasing posteriorly; no lower tail fin fold.

Skull. The elements of the skull are as follows ( Fig. 2 , and Fig. 2 A copied from Peng et al., 2010): upper jaw composed of paired premaxillae at the rostral tip; lateral paired maxillaries; both bear many teeth. Processus dorsalis praemaxillaris extends posteriorly parallel to the longitudinal axis of the skull, and terminates above the anterior ½ of the nasal. Paired nasals situated close to each other anteriorly, reaching anterior of frontal, and extending laterally to the lacrimal. Premaxillary fontanelle present between the premaxillaries and nasals; fronto-parietal fontanelle absent between frontal and parietal; internasal absent between the nasals and premaxillaries. The anterior portion of the lacrimal extends to external nares and orbit; covered by nasal and processus facialis maxillaries. Lacrimal overlaps anterior portion of prefrontal. Paired frontals located in the middle of the skull; overlapped anteriorly by nasals; overlapping anterior portion of parietals. Posterior end of each parietal overlaps exoccipital. Posterior end of the maxillary not sutured to anterior end of pterygoid, but connected to pterygoid by cartilaginous tissue. Squamosal overlaps quadrate and posterior pterygoid; fixed to the lateral area of upper skull ( Fig. 3 A).

Paired vomers lie laterally to the anterior portion of the parasphenoid and form the ventral part of the skull. Anterior part of vomer connected to adjacent premaxilla and maxillary. The posterior vomer overlaps anterior portion of unpaired parasphenoid. Vomerine tooth rows located on posterior portion of the vomer, arranged in shape, not connected at midline. Tooth rows form inner and outer series; inner series beginning at end of vomer and outer series ending at posterior choanae; the inner series (7–8 teeth) slightly longer than the outer series (4–7 teeth). The widest part of parasphenoid located slightly behind prootic. Paired orbitosphenoids enclosed by parasphenoid, frontal and parietal. Operculum posterior to squamosal, shares cartilaginous otic capsule with exoccipital; parasphenoid overlaps the capsule ( Fig. 2 B).

The lower jaw (not shown) consists of four bony elements on each side. The paired dentaries, bearing teeth, enclose Meckel’s cartilage. The long angular lies parallel to dentary and prearticular overlaps angular medially.

Coloration. In life, dorsal color gray-yellow, variegated with yellowish spots, venter grayish, and dorsal tail with distinct yellowish line. Fingers and toes silvery white ( Fig. 1 A). Ventral color generally gray ( Fig. 1 B). In preservative, dorsal color brownish, and ventral color deep grey, yellowish spots appearing as grey spots.

Variation. Internasal bone and premaxillary fontanelle absent in some specimens; premaxillary fontanelle present in all specimens other than holotype. Color varies as follows: CIB-XM3365 brownish with scattered light yellowish spots; CIB-XM3364 and CIB-XM3323 gray-brown, mottled with yellowish spots on dorsum. A yellowish line present on underside of tail of CIB-XM3323 but absent in other specimens.

Larva ( Fig. 3 A; Table 1 ). Head depressed, HW<HL. Lips and eyelids distinct. External gills in three pairs, reddish brown, the inner one longest extending to base of forelimb; outer gill shortest. Limbs slender; fingers and toes flat; no black horny cover at tips. Tail long, TL<SVL; sharpened at tip. Tail fin distinct; dorsal fin originates at midpoint of body, gradually increasing in height posteriorly; and reaching maximum height at midpoint of tail. Ventral fin originates at posterior edge of the cloaca. In life, dorsal color flaxen, mottled with black brownish spots; ventral color gray; tail fin silver white ( Fig. 3 A). In preservative, flaxen becomes brownish, and black brownish spots become yellowish grey; tail fin becomes white.

Egg sacs and eggs. Females produce two egg sacs; joined at the base, forming a sticky handle, attached to under surfaces of stones or wall of cavities. Body of egg sacs cylindrical, largest at midpoint and becoming smaller toward free end, which is silver white, smooth and transparent. Transverse striations on transparent wall of body. Average length of egg sacs (N = 7) is 99.3 mm, ranging from 74.0 to 121.0 mm. Average greatest diameter was 10.1 mm, ranging from 8.9 to 13.0 mm. Average number of eggs in each sac 12, ranging from 13 to 21. Eggs round, diameter from 3.2 to 3.3 mm, eggs arrayed singly or slightly overlapping in sacs ( Fig. 3 B).

Karyotype. The diploid complement consists of 52 chromosomes that are divided into three groups according to their sizes and arm ratios ( Fig. 4 ). The first group is large-sized and biarmed (pairs 1–7), including four metacentric (Nos. 1, 3, 5, and 7), one submetacentric (No. 4), and two subacrocentric (Nos. 2 and 6). The second group is medium-sized and biarmed (pairs 8–13), containing two metacentric (Nos. 12 and 13), three submetacentric (Nos. 8, 9 and 11), and one acrocentric (No. 10). The third group (Nos. 14–26) contains small-sized pairs and all are uniarmed and acrocentric ( Fig. 4 ; Table 2 ). No conspicuous secondary constriction was observed in any of the metaphase plates.

Distribution. Pseudohynobius puxiongensis is known only from the type locality.

Breeding habitat and habits. Shenguozhuang Panda Nature Reserve is located on southeastern Daliangshan Mt. at the eastern edge of the Qinhai-Tibet plateau. The area was deforested for building the Chengdu-Kunming Railway in the 1960s. Today, tree stumps are common. Secondary vegetation is dominated by bamboo ( Fargesia spathacea ) and shrub. The salamanders are always found in the upper parts of small mountain brooks that originate from springs. Maximal height of the brook banks where animals are found is approximately 100 cm, the maximal width not more than 100 cm, and the minimal width less than 50 cm. Dense bamboo and shrub overhang the brook and prevent the penetration of direct sunlight into the habitat of larva. The soil of the bank is loose and scattered with a few caves and rock cavities, which provide shelter for the salamanders. The brooks form many small pools whose depth is less than 20 cm and the clear water moves slowly. The stream bottom contains stones of varying size as well as occasional silt and humus.

Pseudohynobius puxiongensis seems to be a typical terrestrial salamander in that specimens are only found in water during the breeding season. Some local people reported specimens under the roots of bamboo and shrubs not far from the brooks after breeding season. We found an adult male adjacent to a pair of egg sacs ( Fig. 3 C). Females may leave the breeding sites after spawning and males might provide parental care, as in other hynobiid salamanders H. kimurai and H. naevius ( Tago, 1931) , H. nebulosus ( Thorn, 1962) . Larvae in the small stream pools immediately hide under stones when disturbed. Seven egg sacs were discovered; four under large stones, three in soil and stone caves at the brook’s margin.

Breeding season. The breeding season probably lasts from March to the end of April, and perhaps longer. Adult were first found on 20 March 2008 and males were associated with egg sacs on 24–30 April 2008 and on 24 April 2009. Seven pairs of egg sacs were discovered during the last ten days of April 2008 and 2009. They contained embryos of various developmental stages (before stage 20, according to R. Harrison cited from Duellman & Trueb, 1994). During this period, the remnants of snow still covered the mountain slope ( Fig. 3 D). Larvae with external gills were found in November 2008 and 2009.

Resource status and threat. Pseudohynobius puxiongensis was assessed as Data Deficient (Gu et al., 2001). Based on our field survey, the numbers of Ps. puxiongensis appear to be very limited. In October, 2007, four larvae were discovered 2 km away from the type locality. In March, 2008, one adult was discovered at the location where the larvae were found in 2007. In April, 2008, two larvae, four adult males, and three egg sacs were discovered. In April, 2009, four egg sacs were detected. Thus, all Ps. puxiongensis encountered during the three years of field-survey are less than 3 km from the type locality, and adult individuals numbered not more than 200 (unpublished data). Furthermore, the two known localities of Ps. puxiongensis are fragmented and near grazing areas. Local people and animals often use these areas, so that the extent and quality of the habitat continues to decline. As a consequence, we believe that Ps. puxiongensis should be considered a Critically Endangered species according to the criterion B2ab(iii) of IUCN (IUCN, 2001), habitat destruction and degradation caused by human activities is the major threat to Ps. puxiongensis .

No. of chromosome species

m=metacentric, sm=submetacentric, st=subacrocentric, and t=acrocentric. a Data for Ps. flavomaculatus are from Ikebe et al. (2000). b Data for Ps. shuichengensis from Tian et al. (2001). c The centromere position of this chromosome should be t (acrocentric); Tian et al. (2001) represented it as m (metacentric) because they could not distinguish the centromere position.

Remarks. Recently, Li et al. (2010) described Ps. guizhouensis , a new species of Pseudohynobius from Guizhou Province; six species have been recognized in the genus Pseudohynobius . Wei et al. (2009) carefully discussed the differences among four species. Pseudohynobius puxiongensis differs from Ps. flavomaculatus and Ps. guizhouensis by having a distinct yellowish line on the dorsal tail and shorter total length; from Ps. shuichengensis by having yellow dorsal spots; from Ps. kuankuoshuiensis by HL / HW <1.53 and dorsal spots erose; from Ps. jinfo by TL <SVL ( Table 2 ).

The karyotype of Ps. puxiongensis is similar to that of the other two congeners of known karyotype. The diploid chromosome number of Ps. puxiongensis (2n=52) is the same as that of Ps. flavomaculatus ( Ikebe et al., 2000) , but differs from that of Ps. shuichengensis (2n=50; Tian et al., 2001). These three species have similar chromosome groupings. All three species have large-sized biarmed pairs (Nos. 1–7), medium-sized biarmed pairs (Nos. 8–13) and small-sized pairs (Nos. 14–26) although one small pair is missing in Ps. shuichengensis (Nos.14–25). Comparing the karyotype characteristics of these three species, Ps. puxiongensis differs from Ps. flavomaculatus in Nos. 2, 3, 4, 5, 6 and 11, from Ps. shuichengensis in Nos. 3, 4, 6 and 11; and Ps. flavomaculatus from Ps. shuichengensis in Nos. 5 and 6 ( Table 3 ). Regardless, the karyotype of Ps. puxiongensis contains noticeably fewer chromosomes than other hynobiid genera, including Hynobius (2n=56, 58 or 40, Kohno et al., 1991; Qing et al., 2008), Pachyhynobius (2n=64; Yang, 1990), Liua (2n=66; Ikebe et al., 2000), Ranodon (2n=66; Wang et al., 1996), Salamandrella (2n=62, Zeng et al., 1997), Onychodactylus (2n=78; Ikebe et., 1981; Iizuka and Yazawa, 1994) and Batrachuperus (2n=62, 66 or 68; Yang and Zhao, 1988; Yang, 1990; Kuro-o et al., 1998). The karyotypic patterns support the inclusion of Ps. puxiongensis in the genus Pseudohynobius .

The breeding season of Ps. puxiongensis is the same as that of other species in the genus. However, its egg deposition differs from that of Ps. flavomaculatus and Ps. shuichengensis . Nine pairs of egg sacs have been reported for Ps. flavomaculatus and these were found in a stream pond where the spring opening consisted of an earthen top and flagstone bottom. Twelve pairs of egg sacs were detected when digging into the spring entrance, each of which was fixed on the top wall ( Fei and Ye, 1982). Thirty-five egg sacs of Ps. shuichengensis were found from May to June 1994. Like those of Ps. flavomaculatus , egg sacs were attached either on roots or the wall of the spring opening ( Tian et al., 1998). In contrast, the egg sacs of Ps. puxiongensis were not deposited at the opening of a spring.

Figures

FIGURE 1. Dorsal (A) and ventral (B) views of Pseudohynobius puxiongensis (CIB-XM 3364).

FIGURE 2. Diagrammatic drawing of skull of Pseudohynobius puxiongensis (CIB-XM 3323) in dorsal (A) and ventral (B) views with dermal bones in white or white with black dots (in deeper zones of the skull), cartilage in gray, teeth in open circles, and gaps or holes in black, where pm = premaxillary, n = nasal, pf = prefrontal, l = lacrimal, m = maxillary, f = frontal, os = orbitosphenoid, p = parietal, po = prootic, pt = pterygoid, q = quadrate, s = squamosal, o = operculum, ex = exoccipital, v = vomer, ps = parasphenoid. Figure 2 A cited from Peng et al. (2010).

FIGURE 3. The larva (A, CIB-XM 3198), egg sac (B), a male staying by a pair of egg sacs (C), and the habitat (D) of Pseudohynobius puxiongensis.

FIGURE 4. The karyotype of Pseudohynobius puxiongensis.

Tables

TABLE 2. Character comparison among six species of the genus Pseudohynobius.

Vomerine teeth	11–15 12–17	12–17
Total length of female Total length of male Tail length to snout-vent length ratio Head length to head width ratio Dorsal colour in life Spots on dorsal tail	NA 138.0–180.0 108.5–137.5 158.0–189.0 <1 <1 <1.55 <1.55 gray-yellow, mottled with grayish brown, mottled with yellowish spots larger yellowish spots yellowish spots in a line irregular yellowish spots	150.4–154.7 NA <1>1.55 grayish brown, mottled with oval yellowish spots oval yellowish spots
continued.
Traits Vomerine teeth Total length of female Total length of male	Ps. shuichengensis 3 Ps. jinfo 4 8–14 8–9 182.5–213.2 163.3 177.7–209.9 198.7	Ps. guizhouensis 5 12–17 157.1–203.4 176.0–184.0
Tail length to snout-vent length ratio	<1>1	<1
Head length to head width ratio	>1.55 <1.55	>1.55
Dorsal colour in life	purplish brown without grayish brown, more or less mot- spots tled with smaller yellowish spots	purplish brown, mottled with orange or yellowish circular spots
Spots on dorsal tail	Without spots small yellowish spots	orange or yellowish circular spots

TABLE 1. Measurement data of Pseudohynobius puxiongensis. All measurements are in mm, see text for abbreviation. CIB 65 II 0220 CIB-XM 3364 CIB-XM 3323 CIB-XM 3365 CIB-XM 3198 Adult male Adult male Adult male Adult male Larva

TOL 133.0 SVL 71.4 HL 16.8 HW 11.3 SL 3.5	136.7 67.7 16.4 11.5 3.3	108.5 62.7 15.4 10.1 3.2	137.5 69.8 16.5 13.1 3.3	63.1 33.1 9.2 8.2 2.4
TRL 51.0 VL 3.9	51.4 4.0	47.1 3.7	53.3 4.1	24.0 1.6
IOS 3.3 DE 4.4 TL 61.6 TH 5.7	3.7 3.3 60.6 6.2	2.8 3.2 47.3 5.3	3.7 3.5 63.6 6.2	2.1 2.3 28.5 5.9
TW 7.4 FLL 19.7 HLL 20.8 AGS 31.5	6.9 19.4 19.4 30.2	4.8 17.1 17.1 27.3	7.1 19.5 20.6 33.6	3.3 6.8 9.6 16.0

TABLE 3. Centromere positions comparison of chromosomes in three species of Pseudohynobius (Ps. flavomaculatus, Ps. shuichengensis and Ps. puxiongensis).

	Ps. flavomaculatus a	Ps. shuichengensis b	Ps. puxiongensis
1	m	m	m
2	m	m	st
3	sm	sm	m
4	st	st	sm
5	st	m	m
6	m	sm	st
7	m	m	m
8	sm	sm	sm
9	m	sm	sm
10	t	t	t
11	m	m	sm
12	m	m	m
13	m	m	m
14-25	t	t c	t
26	t	NA	t