The Dorsal Tail Tubercle of Mertensiella Caucasica and M. Luschani (Amphibia: Salamandridae)

JOURNAL OF MORPHOLOGY 232:93–105 (1997)

The Dorsal Tail Tubercle of Mertensiella caucasica and M. luschani (Amphibia: Salamandridae)

DAVID M. SEVER,1* MAX SPARREBOOM,2 AND GUENTHER SCHULTSCHIK3 1Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556 22597 SC Den Haag, The Netherlands 3Sechsschimmelgasse 7/19, A-1090 Wien, Austria

ABSTRACT Males of the two species of Mertensiella (M. caucasica and M. luschani) possess a tubercle projecting from the skin of the dorsal tail base, the single morphological character that deﬁnes the genus. The dorsal tail tubercle functions during courtship, and its role is similar in both species. The tubercle is inserted into the cloaca of the female during ventral amplexus, shortly before the male deposits a spermatophore. Histological examination, however, revealed that the dorsal tubercles differ structurally between the two species. In M. caucasica, the tubercle consists primarily of elongate mucous glands, with granular glands occurring only at the base. Both mucous and granular glands of the tubercle are larger than those in typical skin. Unlike typical skin, however, mucous glands are larger than granular glands. In M. luschani, mucous glands and granular glands occur throughout the tubercle, and the granular glands are larger than the mucous glands, although both types are larger than those in typical skin. The dorsal tubercles of M. caucasica and M. luschani may not be homologous structures and may have resulted from convergent evolution. J Morphol 232:93–105, 1997. r 1997 Wiley-Liss, Inc.

Mertensiella contains two species, M. cau- regarding courtship behaviour and phylog- casica, found in Turkey bordering the south- eny of Mertensiella. eastern edge of the Black Sea and in west Georgia, and M. luschani, occurring along MATERIALS AND METHODS the Mediterranean coast of southwestern Three Mertensiella luschani (2 males, 1 Turkey and on nearby Greek islands female) and M. caucasica (1 male, 2 females) (Griffiths, ’96). Mertensiella caucasica is an utilized in this study were from the verte- oviparous species whose breeding is depen- brate collection of the Naturhistorisches Mu- dent upon water (Schultschik, ’94a; Tark- seum Wien (NMW), Wien (Vienna), Austria. hnishvili, ’94), and M. luschani is a vivipa- The M. luschani (NMW 23221) were col- rous form that breeds entirely on land (O¨ zeti, lected between March 31 and April 2, 1978, ’79; Rehberg, ’81; Polymeni, ’94). on the Greek island of Karpathos, and are Males of Mertensiella are characterized by the subspecies M. I. helverseni (Baran and ¨ the presence of a conspicuous tubercle pro- Ucu¨ ncu¨ , ’94). The M. caucasica (NMW 19184) jecting from the skin of the dorsal surface of were collected July 23, 1968, in Bayburt, the tail base; this structure is autapomor- Turkey. In addition, four M. caucasica (2 phic for Mertensiella (O¨ zeti, ’67). Schubert males, 2 females) were loaned to us by J.W. (’74) studied the fine structure and histo- Arntzen (University of Wales, Bangor, UK) chemistry of the dorsal tail tubercle of and were designated JWA 1–4. These speci- M. caucasica, but the microscopic anatomy mens were collected from the Nedzura River, Borjomi Canyon, in central Georgia (date of the dorsal tail tubercle of M. luschani is unknown). described here for the first time. We report differences in the histology of the dorsal tail tubercles of M. caucasica and M. luschani, *Correspondence to: Dr. David M. Sever, Department of Biol- and consider the implications of our findings ogy, Saint Mary’s College, Notre Dame, IN 46556. r 1997 WILEY-LISS, INC. 94 D.M. SEVER ET AL.

The specimens initially were fixed in 10% licles 2.0- to 2.9-mm diameter (mean 5 2.6, formalin and subsequently stored in 60% SE 5 0.09), five small, pigmented follicles ethanol. Snout-vent length (SVL) was mea- 1.2- to 1.4-mm diameter (mean 5 1.3, sured from tip of the snout to posterior end SE 5 0.03), and approximately 200 small, of the vent. Testes, vasa deferentia, and the white follicles 0.4- to 1.0-mm diameter dorsal tubercle with several millimeters of (mean 5 0.7, SE 5 0.05). One of the female surrounding skin were removed from males. M. caucasica (JWA 3, 72.6 mm SVL) from From females, a patch of skin was excised Georgia (collection date unknown) also con- from the area corresponding to that occu- tains hypertrophied oviducts and three size pied by the caudal tubercle of males. The classes of follicles: 21 large pigmented fol- tissue was dehydrated in a graded series of licles 2.0- to 3.1 mm-diameter (mean 5 2.7, alcohol, cleared with toluene, and embedded SE 5 0.10), 12 small, pigmented follicles 1.2- in paraffin. Transverse or sagittal sections to 1.8-mm diameter (mean 5 1.5, SE 5 0.05), were cut at 10 µm with a rotary microtome, and 260–300 small, white follicles 0.4- to affixed to albuminized slides, and alternate 0.8-mm diameter (mean 5 0.6, SE 5 0.04). slides stained with hematoxylin and eosin The other female (JWA 4, 72.4 mm SVL) (H&E) (general cytology), the periodic acid- from Georgia possesses two size classes of Schiff procedure (PAS, for neutral carbohy- follicles: 21 pigmented follicles 1.0- to 1.6-mm drates) counterstained with Alcian blue diameter (mean 5 1.3, SE 5 0.05) and 200– 8GX at pH 2.5 (AB, for carboxylated gly- 240 white follicles 0.4- to 0.7-mm diameter cosaminoglycans), and brilliant blue B (BB, (mean 5 0.5, SE 5 0.03). for proteins). Staining procedures followed Kiernan (’90). Maximum diameters of histological sections of glands were measured Mertensiella luschani with an ocular micrometer in a light micro- The males and female collected between scope at 3100. March 31 and April 2 on Karpathos are in Ovaries were removed from females, and the post-mating period, as indicated by the the numbers of vitellogenic follicles were condition of the testes and spermathecae. In counted. Measurements of 11 follicles of the the males (NMW 23221.2, 67.8-mm SVL and same developmental stage were measured NMW 23221.10, 63.3-mm SVL), some sperm with an ocular micrometer in a dissecting remain in anterior testicular lobules, but microscope at 320. Statistical analyses used most lobules are evacuated of sperm and con- SYSTAT-5 for Windows, Systat (Evanston, IL). tain only spermatogonia (Fig. 2C). The vasa RESULTS deferentia are filled with sperm (Fig. 2C). Reproductive condition of the samples A female (NMW 23221.20, 77.0-mm SVL) Mertensiella caucasica contains 350–400 yellowish follicles 0.6- to 1.45-mm diameter (mean 5 1.05, SE 5 0.08) The males and females show indications plus scattered small white follicles 0.2- to of recent breeding. The testes of a male 0.4-mm diameter (mean 5 0.3, SE 5 0.01). Mertensiella caucasica (NMW 19184.8, The oviducts are narrow and straight. Sper- 65.6-mm SVL) collected July 23 in Turkey mathecae contain numerous sperm (Fig. 2D). are evacuated of sperm and contain only Thus, this specimen of Mertensiella luschani spermatogonia, with some proliferation ap- apparently mated recently but is not in con- parent in posterior lobules; sperm are ab- dition for ovulation and fertilization of eggs. sent in the reproductive ducts as well. Both males from Georgia (JAW 1, 71.6-mm and Dorsal tail skin of females JAW 2, 69.8-mm SVL; collection date unknown) have some sperm remaining in the The epidermis of the skin of the dorsal tail anterior ends of the multiple-lobed testes base in females of both Mertensiella cauca- (Fig. 2A) and in the reproductive ducts. sica and M. luschani is typical of amphibian A female Mertensiella caucasica (NMW skin, consisting of three to four layers of 19184.9, 71.8-mm SVL) collected in Turkey epithelial cells deep to a superficial keratin- lacks oviducal eggs, but the oviducts are ized layer (Fig. 3). Opening onto the epider- hypertrophied (relatively widened and con- mal surface are simple, acinar, exocrine voluted). The spermathecae contain some glands of two types: granular glands and sperm, but many of the tubules are empty mucous glands. The multicellular acini of (Fig. 2B). Three classes of follicles are pre- the mucous and granular glands are in the sent in the ovaries: 24 large, pigmented fol- loose connective tissue portion of the dermis, MERTENSIELLA TAIL TUBERCLE 95 Figures 4 and 5, respectively, and midsagittal sections are shown in Figure 6. The epidermis, dermis, mucous glands, and granular glands in the area surrounding the tubercle have the same anatomy and relative size as described for females (Figs. 3–6). These four components differ from the typical amphibian condition in the dorsal tubercles, and interspecific differences occur between Mertensiella caucasica and M. luschani in the anatomy of the dermis and of the two gland types. The epidermis of the dorsal tubercle, however, is similar in both species. The epider- Fig. 1. Mertensiella, showing external appearance of mis varies from 8–10 layers thick (Figs. 4–6), the dorsal tail tubercle. A: Mertensiella caucasica (NMW 19184.8, 65.6-mm SVL) collected July 23 in Turkey. and the outer 2–3 layers are keratinized. B: Mertensiella luschani (NMW 23221.2, 67.8-mm SVL) Gaps between the most superficial layers collected between March 31 and April 2 on Karpathos. indicate that the keratinized layers are Dt, dorsal tail tubercle. readily sloughed. The stratum compactum of the dermis continues ventral to the pa- pilla, but is relatively thicker than in the the stratum spongiosum. Deep to the stra- pre- or post-tubercular regions. Deep to the tum spongiosum are thick layers of collagen collagenous stratum compactum are bands fibers that form the stratum compactum of of longitudinal smooth muscle and deeper the dermis, which separates the skin from still are skeletal muscle bundles of the tail deeper skeletal muscles (Houck and Sever, myotomes (Figs. 4–6). Neural ganglia occur ’94). ventral to the dorsal tubercle and are associ- The granular and mucous glands in fe- ated with middorsal portions of the skeletal males are not hypertrophied to form any muscle bundles in Mertensiella luschani (Fig. swelling distinguishing the dorsal tail base 5B). from other regions of the skin. The mucous The continuation of the stratum compac- glands are simple acinar glands that are tum from adjacent portions of the skin forms PAS1 and AB1 for neutral and acidic mu- the connective tissue matrix of the tubercle cosubstances, and are BB2 for proteins. The (Figs. 4–6). This tissue is more fibrous than granular glands are widest in their antero- the stratum spongiosum in typical skin. The posterior dimension and are PAS1,BB1, fibers of the stratum spongiosum of the dor- and AB2. In a female Mertensiella cauca- sal tubercle, however, are irregular, in con- sica (NMW 19184.9), the mucous glands are trast to the regular bands of the stratum 80- to 120-µm diameter (mean 5 100.1, compactum. The stratum spongiosum of the SE 5 3.10) and the granular glands are 300- dorsal tubercle of Mertensiella caucasica is to 450-µm diameter (mean 5 363.6, more pigmented than that of M. luschani. SE 5 13.3). In a female M. luschani (NMW Other differences between the dorsal tu- 23221.20), the mucous glands are 80- to bercles of Mertensiella caucasica and M. lus- 140-µm diameter (mean 5 110.0, SE 5 7.00) chani involve the anatomy of the mucous and granular glands are 250- to 360-µm and granular glands. In the following sec- diameter (mean 5 288.2, SE 5 9.98). tions, some measurements of the dorsal tu- Dorsal tail tubercle of males bercles and glands in the two species are given, and the differences in the glands are In both Mertensiella caucasica and M. lus- described. chani, the dorsal tail tubercles are situated middorsally with the anterior end of the tubercle in the same transverse plane as the Mertensiella caucasica posterior edge of the cloacal orifice. The cau- In a 65.6-mm SVL male from Turkey dal tubercles are bent anteriorly, and the (NMW 19184.8), the caudal tubercle is 2.1 apex is knob-like in M. caucasica and more mm in height and lengths are 2.2 mm at the pointed in M. luschani (Fig. 1). Transverse base and 1.6 mm at the apex. The males sections through the caudal tubercles of from Georgia (JWA-1, 71.6-mm SVL and M. caucasica and M. luschani are shown in JWA-2, 69.8-mm SVL) have caudal tubercles 96 D.M. SEVER ET AL.

Fig. 2. Mertensiella. Paraffin sections stained with between March 31 and April 2 on Karpathos. D: Trans- hematoxylin and eosin. A: Sagittal section through a verse section through the spermathecae of a female testis of a male M. caucasica (JWA 1, 71.6-mm SVL) M. luschani (NMW 23221.20, 77.0-mm SVL) collected from Georgia. B: Transverse section through the sper- between March 31 and April 2 on Karpathos. Ct, cloacal mathecae of a female M. caucasica (NMW 19184.9, tube; El, evacuated lobules; Se, Sertoli cells; Sg, sper- 71.8-mm SVL) collected July 23 in Turkey. C: Sagittal matogonia; Sp, sperm; St, spermathecae; Ts, testis; Vd, section through a testis and vas deferens of a male vas deferens. M. luschani (NMW 23221.2, 67.8-mm SVL) collected MERTENSIELLA TAIL TUBERCLE 97

Fig. 3. Mertensiella. Transverse sections showing and April 2 on Karpathos. Ad, adipose cells; Ep, epider- skin glands of the dorsal tail base of females. A: Merten- mis; Gg, granular gland; Mg, mucous gland; Sc, stratum siella caucasica (NMW 19184.9, 71.8-mm SVL) collected compactum; Sm, skeletal muscle; Ss, stratum spongio- July 23 in Turkey. B: Mertensiella luschani (NMW sum. 23221.20, 77.0-mm SVL) collected between March 31 98 D.M. SEVER ET AL.

Fig. 4. Male Mertensiella caucasica (JWA1, 71.6-mm bercle. D: Posterior portion of the tubercle. E: Integu- SVL) from Georgia. Transverse sections through the ment just posterior to the tubercle. Ad, adipose cell; Ep, dorsal tail tubercle area stained with hematoxylin and epidermis; Gg, granular gland; Mg, mucous gland; Sc, eosin. A: Integument just anterior to the tubercle. B: stratum compactum; Sm, skeletal muscle; Ss, stratum Anterior portion of the tubercle. C: Middle of the tu- spongiosum. MERTENSIELLA TAIL TUBERCLE 99

Fig. 5. Male Mertensiella luschani (NMW 23221.10, D: Posterior portion of the tubercle. E: Integument just 63.3-mm SVL) collected between March 31 and April 2 posterior to the tubercle. Ep, epidermis; Gg, granular on Karpathos. Transverse sections through the dorsal gland; Mg, mucous gland; Ng, neural ganglia; Sc, stra- tail tubercle area stained with hematoxylin and eosin. tum compactum; Sm, skeletal muscle; Ss, stratum spon- A: Integument just anterior to the tubercle. B: Anterior giosum. portion of the tubercle. C: Middle of the tubercle. 100 D.M. SEVER ET AL.

Fig. 6. Mertensiella. Midsagittal sections through 67.8-mm SVL) collected between March 31 and April 2 the dorsal tail tubercles stained with hematoxylin and on Karpathos. Ep, epidermis; Gg, granular gland; Mg, eosin. Anterior is towards the right. A: Mertensiella mucous gland; Sc, stratum compactum; Sm, skeletal caucasica (NMW 19184.8, 65.6-mm SVL) collected July muscle; Ss, stratum spongiosum. 23 in Turkey. B: Mertensiella luschani (NMW 23221.2, MERTENSIELLA TAIL TUBERCLE 101 2.3 mm and 1.8 mm in height, and 3.5 mm of the dorsal tubercle are larger than in the and 2.1 mm at the base, respectively. surrounding skin, and, as elsewhere, the Both granular and mucous glands occur in granular glands are larger than the mucous the dorsal tubercle of Mertensiella cauca- glands (Table 1). sica, but the granular glands are limited to the basal regions of the tubercle (Figs. 4, 6). Comparison of Mertensiella caucasica and Elongate mucous glands, however, secrete Mertensiella luschani onto the walls of the entire tubercle dorsal to the basal area of granular glands. These A three-way factorial analysis of variance mucous glands are tubular, and their elon- was conducted among maximal gland dimen- gate, distal portions curve ventrally and oc- sions in the dorsal tubercle and surrounding cupy much of the dermal area of the dorsal areas, using species (Mertensiella caucasica tubercle (Figs. 4, 6). Unlike other areas of and M. luschani), area (pre-tubercle, tu- the skin, the mucous glands in the dorsal bercle, and post-tubercle), and gland type tubercle of M. caucasica are larger in their (mucous or granular glands) as factors. The longest dimension than the granular glands. analysis reveals highly significant differ- The granular glands of the dorsal tubercle, ences at all levels except the gland type-area however, are larger than those in the sur- interaction (Table 1). The three-way interac- rounding skin (Table 1). tion is depicted in Figure 7. The relation- Mertensiella luschani ships between gland type and area are the same in areas 1 (pre-tubercle) and 3 (post- NMW 23221.2 (67.8-mm SVL) has a caudal tubercle 2.3 mm in height, and lengths tubercle) in both species (i.e., granular glands are 2.9 mm at the base and 1.5 mm at the appear significantly larger than mucous apex. For NMW 23221.10 (63.3-mm SVL), glands in each of these areas). However, for the caudal tubercle is 2.0 mm in height, 2.1 area 2, granular glands are larger in mm long at the base, and 1.4 mm long at the M. luschani, but mucous glands are larger in apex. The dorsal tail tubercle of Mertensiella M. caucasica. Because of this interaction, luschani contains both mucous glands and the other significant effects (main effects granular glands along all of its surfaces, and two-way interactions) must be evalu- although few glands of either type occur ated with caution. That is, these effects can along the apex of the middorsal ridge (Figs. only be evaluated in the context of one spe- 5, 6). Both the granular and mucous glands cies or the other.

TABLE 1. Three-way factorial analysis of variance of gland diameters from the dorsal tubercle of Mertensiella caucasica and M. luschani1 Mucous glands Granular glands Species/ region Range Mean SE Range Mean SE M. caucasica Pre-tubercle 90–180 102.7 2.56 160–320 230.5 8.79 Tubercle 250–660 588.2 56.10 350–740 472.3 21.40 Post-tubercle 90–130 101.4 3.62 170–330 213.2 11.00 M. luschani Pre-tubercle 90–140 106.4 2.68 120–260 190.5 9.86 Tubercle 110–220 146.8 6.15 310–510 380.9 10.90 Post-tubercle 90–130 109.5 2.03 110–240 166.4 6.36

Analysis of variance Source Sum-of-squares DF Mean-square F-ratio P Species 677109.470 1 677109.470 89.559 0.000 Area 3511082.576 2 1755541.288 232.199 0.000 Type 455836.742 1 455836.742 60.292 0.000 Species*area 899264.394 2 449632.197 59.471 0.000 Species*type 115836.742 1 115836.742 15.321 0.000 Area*type 24159.848 2 12079.924 1.598 0.204 Species*area*type 585023.485 2 292511.742 38.689 0.000 Error 1905250.000 252 7560.516

1Measurements are in µm; N 5 22 for each gland type. 102 D.M. SEVER ET AL. tia of males and in the spermathecae of females. Several size classes of follicles were found in the ovaries of M. caucasica. Posses- sion of developing follicles of different stages may increase opportunity to oviposit when conditions become most favorable (Sharon, ’95; M.R. Warburg, personal communica- tion). The female of viviparous M. luschani examined had abundant sperm in the spermathecae, but lacked large ovarian follicles or convoluted oviducts. A considerable period of sperm storage is implied. The question of the purpose of the dorsal tail tubercle has intrigued observers since the beginning of the century. It was com- monly believed that the function of this structure was to stimulate the female during courtship (Knoblauch, ’05; Lantz, ’11; Wolter- storff et al., ’36; Herre, ’52; Obst and Rotter, ’62; Arnold, ’87), but until observations of the entire courtship sequence were avail- able, this was not confirmed. The full reproductive sequence, from the beginning of courtship behavior to the transfer of the spermatophore, was described by Rehberg (’81) and Klewen (’88) for Mertensiella luschani and by Schultschik (’94a) for M. caucasica. Although differences exist between the sexual behavior of both species, with Fig. 7. Mertensiella caucasica (A) and M. luschani M. luschani mating on land exclusively and (B). Relationships between mean gland size of mucous M. caucasica showing first an aquatic and glands (Mg) and granular glands (Gg) and the area in which the glands occur: (1) pre-tubercle, (2) tubercle, (3) then a terrestrial phase in the mating se- post-tubercle. SEM, standard error of the mean. quence, the use of the caudal tubercle is similar, and occurs on land. During ventral amplexus, when the male is carrying the DISCUSSION female on his back with his forelimbs hooked The dorsal tail tubercle is a characteristic over hers, the pair makes body movements feature in males of the two species of Merten- during which the male rubs the caudal tu- siella and is unique among urodeles. Baso- bercle on the cloacal orifice of the female and glu and Atatur (’75) and Basoglu and Baran eventually inserts it. This takes place shortly (’76), however, reported the occurrence of a before the male deposits a spermatophore slight elevation on the tail of females of (Rehberg, ’81; Schultschik, ’94a). M. luschani in the area where the tubercle The caudal tubercle, therefore, serves a occurs in males. We failed to find any such similar role in both species of Mertensiella. swelling resulting from the hypertrophy of By rubbing the tubercle on the female’s ven- skin glands in females of either M. caucasica ter, the male apparently stimulates the fe- or M. luschani. male during the first stage of amplexus. The occurrence of the dorsal tubercle is Related species (Salamandra, Chioglossa), independent of breeding condition (Klewen, which lack the caudal tubercle, make very ’88), although seasonal variation in histol- similar movements (‘‘body shifting’’) during ogy of the tubercle has not been studied. ventral amplexus (Himstedt, ’65; Joly, ’66; Also, the durations of the breeding seasons Ha¨feli, ’71; Arnold, ’87; Andreone, ’92). A of Mertensiella caucasica and M. luschani caudal tubercle may enhance the effect of have not been definitely established. The such rubbing movements, but the behavior specimens we examined were in breeding or pattern by itself is not dependent on pres- post-mating condition as determined by presence of such a structure. The rubbing move- ence of sperm in the testes and vasa deferen- ments may be a necessary preliminary be- MERTENSIELLA TAIL TUBERCLE 103 havior to make the female responsive before glands are derived from one of these types or the caudal tubercle can be inserted. The originated de novo. The ancestral function of pseudo-coitus that follows may serve to as- mucous glands likely involved water certain that the female is ready for sperm economy, serving as a lubricant in water and transfer. In M. caucasica the caudal tubercle keeping the skin moist on land (Noble, ’31; is approximately the size of a spermato- Habermehl, ’94). The ancestral function of phore and could function as a ‘‘pseudo- granular glands is unclear. Currently, the spermatophore,’’ which is inserted and then glands produce a variety of bioactive pep- withdrawn when the female is about to pick tides, steroidal alkaloids, and their deriva- up the real spermatophore (Arnold, ’87). The tives (Barthalmus, ’94; Erspamer, ’94), which apparent effect of the pseudo-coitus is that is have been found to serve as toxins (Daly stabilizes the female precisely over the spot et al., ’78; Erspamer, ’94), antifungal and where the spermatophore will be dropped antibacterial agents (Habermehl and and can be picked up successfully (Sch- Preusser, ’69; Bettin and Greven, ’86), and in ultschik, ’94a). Apart from a mechanical nutrient storage (Williams and Larsen, ’86). stimulus and maneuvering device, the cau- The secretions of the mucous glands of the dal tubercle may additionally provide a spe- dorsal tubercle could serve a lubricating cific chemical stimulus, since it contains function for insertion of the structure into glands with specific types of secretion. the female cloaca. The additional occurrence Schubert (’74), in a cytological study of the of granular glands along the entire border of dorsal tubercle of Mertensiella caucasica, re- the dorsal tubercle of Mertensiella luschani ported that ‘‘the glands appear to be of one may relate to its completely terrestrial life- type only . . . ovoid or tubular glands of large style; courtship and mating occurs entirely size’’ whose cytoplasm is similar to typical on land in this viviparous species (Rehberg, mucous glands in the skin. Besides shape ’81). Licht and Sever (’93) noted that the and size, Schubert (’74) found that the mu- granular gland secretions from the parotoid cous glands of the dorsal tubercle differ from gland of Ambystoma gracile (Ambystomati- those in the rest of the skin in the ultrastruc- dae) are insoluble in water. Mertensiella cau- ture of their secretory granules, suggesting casica initiates courtship in the water, so the that normal skin glands and those in the female’s cloaca and the male’s dorsal tu- tubercle produce chemically different secre- bercle (containing only mucous glands, ex- tions. Our specimens were unsuitable for cept at the base) have been in contact with examination by electron microscopy, but light water, although insertion of the spike occurs microscopy confirms Schubert’s observation on land (Schultschik, ’94a,b). Epidermal ru- that much of the dorsal tubercle is composed gosities on the dorsum of the male help of mucous glands that are larger and more prevent slippage of the female while the elongate than those in the rest of the skin. In male positions her for insertion of the caudal addition, we found granular glands in the tubercle. The female deposits eggs in water base of the dorsal tubercle of M. caucasica, 2 or 3 days after mating (Tarkhnishvili, ’94), and these granular glands also are larger or even 2 months after mating (Schultschik, than those in the surrounding skin, al- ’94b). though smaller than the mucous glands of The glands in the dorsal tubercle also could the dorsal tubercle (Table 1). release courtship pheromones. Courtship Mertensiella luschani has mucous glands glands in the skin of salamanders that have interspersed with granular glands along the received the most careful study are the ge- walls of the dorsal tubercle, similar to the nial glands posterior to the eyes of Notoph- anatomy found in typical skin. The only thalmus viridescens (Salamandridae) and difference is that the mucous and granular glands on the chin (mental glands) and dor- glands in the dorsal tubercle are larger than sal tail base (caudal glands) in male Pleth- those of the surrounding skin, although the odontidae (Houck and Sever, ’94). The genial same relationship holds (mucous glands are glands and mental glands both appear to be smaller than granular glands). modified mucous glands (Sever, ’76; Pool and Houck and Sever (’94) recently discussed Dent, ’77) whereas the caudal courtship the evolution of the amphibian integument. glands are modified granular glands (Sever, The presence of typical mucous glands and ’89). However, the cloaca of female salaman- granular glands in the skin is a synapomor- drids is not particularly suited for absorp- phy for lissamphibians, and any additional tion of pheromones, as much of the cloaca is 104 D.M. SEVER ET AL. lined with keratinized epidermis, and the of specimens. We are grateful to R. Jensen cavity is not highly vascularized (Sever, ’92). for help with the statistical analyses and O¨ zeti (’67) compared a number of morpho- preparation of Figure 7. We thank W. Kloas logical characters of Mertensiella caucasica, for aid in obtaining copies of some papers. M. luschani, and Salamandra salamandra. She found that M. luschani appears more LITERATURE CITED similar in many characters to S. salaman- Andreone, F. (1992) Observations on the territorial and dra than to M. caucasica. Indeed, she found reproductive behaviour of Salamandra lanzai and that the presence of the dorsal tubercle was considerations about its protection (Amphibia: Sala- the sole character defining the genus Merten- mandridae). Brit. Herp. Soc. Bull. 39:31–33. siella, and recommended relegating Merten- Arnold, S.J. (1987) The comparative ethology of courtship in salamandrid salamanders. I. Salamandra and siella to a subgenus within Salamandra. Chioglossa. Ethology 74:133–145. Titus and Larson (’95) performed phyloge- Baran, I., and S. U¨ cu¨ ncu¨ (1994) The state of Mertensi- netic analyses of salamandrid relationships ella luschani in Turkey. Mertensiella 4:33–40. using both morphological and molecular Barthalmus, G. (1994) Biological roles of amphibian skin. In H. Heatwole and G.T. Barthalmus (eds): Am- data. Maximum parsimony analyses using a phibian Biology. Vol. 1. The Integument. Chipping combination of morphological and molecular Norton, NSW, Australia: Surrey Beatty and Sons, data and using molecular data alone failed pp. 382–410. to support monophyly of Mertensiella. The Basoglu, M., and M. Atatur (1975) A new population of the Lycian salamander, Mertensiella luschani (Stein- maximum parsimony trees place M. lus- dachner) from Finike in southwestern Anatolia. Istan- chani with Salamandra and M. caucasica bul Univ. Fen. Fak. Mec. Ser. B 40:89–93. with Chioglossa, making Mertensiella para- Basoglu, M., and T. Baran (1976) The subspecific status phyletic with respect to other ‘‘true’’ sala- of the population of Mertensiella luschani (Stein- dachner) in the Antalya region of southwestern Anato- manders (Chioglossa, Mertensiella, and Sala- lia. Ege Univ. Fen. Fak. Ilmi Raporlar Ser. 235:3–13. mandra). Bettin, C.H., and H. Greven (1986) Bacteria on the skin Our finding that the dorsal tubercles of of Salamandra salamandra (L.) (Amphibia, Urodela) Mertensiella caucasica and M. luschani dif- with notes on their possible significance. Zool. Anz. 216:267–270. fer anatomically raises the possibility of con- Daly, J.W., G.B. Brown, and M. Mensah-Dwumah (1978) vergence in their evolution. One cannot, how- Classification of skin alkaloids from neotropical poi- ever, rule out the possibility that a caudal son dart frogs (Dendrobatidae). Toxicon 16:163–188. tail tubercle occurred in the common ances- Erspamer, V. (1994) Bioactive secretions of the integument. In H. Heatwole and G.T. Barthalmus (eds): tor of M. caucasica and M. luschani and Amphibian Biology. Vol. 1. The Integument. Chipping subsequently evolved the interspecific differ- Norton, NSW, Australia: Surrey Beatty and Sons, ences we found. pp. 178–350. In summary, we found that the dorsal tail Griffiths, R.A. (1996) Newts and Salamanders of Europe. San Diego: Academic Press. tubercles of Mertensiella caucasica and Habermehl, G. (1994) The biological relevance of Sala- M. luschani differ structurally and therefore mandra venom. Mertensiella 4:209–214. may have different functional properties as Habermehl, G., and H.J. Preusser (1969) Hemmung des well. Obtaining specimens of these species Wachstums von Pilzen und Bakterien durch das Haut- drusensekret von Salamandra maculosa. Z. Naturfor- for histological study was difficult, and our sch. 24b:1599–1601. samples were small. Intraspecific variation Ha¨feli, H.P.(1971) Zur Fortpflanzungsbiologie des Alpen- in anatomy of species-specific sexually dimor- salamanders (Salamandra atra Laur.). Rev. Suisse phic glands, however, has been found to be Zool. 78:235–293. Herre, W. (1952) Beitra¨ge zur Kenntnis des Sexualdimor- minimal when larger samples have been phismus einiger Salamandriden. Zool. Anz. 148:215– analyzed (Sever, ’94), so we believe that dif- 231. ferences we found in structure between the Himstedt, W. 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