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 histo- logical 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 un- known) 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 midsagit- tal sections are shown in Figure 6. The epidermis, dermis, mucous glands, and granular glands in the area surround- ing 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. lus- chani in the anatomy of the dermis and of the two gland types. The epidermis of the dorsal tubercle, how- ever, 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.