unsuitable for oviposition by S. albicans. However, no significant Herpetological Review, 2006,37(2), 161-165. negative relationship was recorded. (0 2006 by Society for the Study of Amphibians and The patterns found show aggregation size to be remarkably con- stant by the standards of any vertebrate populations. The perma- Abundance and Biomass of Twelve of nence of males in aggregation was high, and the little variation in Native to Northeastern Kansas aggregation size was explained by variations in the numbers of males entering the aggregation each month. The possibilility that HENRY S. FITCH aggregation size could be used as in index to population size is University of Kansas Fitch Natural History Reservation jeopardized by its high constancy and lack of relationship with 2060 East 1600 Road, Lawrence, Kansas 66044, USA rainfall patterns. Possibly aggregation size is rather controlled by social factors regulating the number of males calling at a time in and ALICE F. ECHELLE any given place. Department of Zoology, Oklahoma State University Stillwater, Oklahoma 74078, USA Acknowledgments. —We thank IBAMA (Institute Brasileiro de Meio e-mail: [email protected] Ambiente e Recursos Naturais Renovaveis) for allowing the work at Parque Nacional da Serra dos Orgaos; Institute Nacional de Meteorologia The purposes of this paper are to examine abundance and biom- (INEMET) for climatic data; Sergio Potsch de Carvalho e Silva for teach- ass estimates of the dozen most abundant species in our ing on amphibians, for logistic support and co-supervising DN's MSc area of northeastern Kansas, and to relate these numbers to infor- dissertation; the several colleagues, especially Fabio Nascimento, who mation reported in the literature. Hirth and King (1968) stated, "In helped in field work; Romulo Barroso for company in all field work and spite of all the current interest in ecosystem ecology there is still a discussions throughout the study; Jose P. Pombal Jr., William Magnusson, dearth of information concerning biomass densities of snakes in Jon Loman, Monique Van Sluys, Ronaldo Fernandes, Gunther Koehler and an anonymous referee for very useful comments on the manuscript; various habitats." Except for a few studies (e.g., Bonnet et al. 2002; Jorge Luiz do Nascimento for ideas in this study and for financial sup- Godley 1980; Hirth and King 1968; Reichenbach and Dalrymple port. 1986; Winne et al. 2005), the situation does not seem to have changed appreciably in the last 37 years. LITERATURE CITED METHODS AND STUDY AREA BOKERMANN, W. C. A. 1967. Dos nuevas especies de Hyla del grupo catharinae (Amphibia, Hylidae). Neotropica 13:62-66. Snakes have been studied for the past 56 years on Kansas DEAN, W. 1996. A Ferro e Fogo – A Historia e a Devastacao da Mata Atlantica Brasileira. Companhia das Letras, Sao Paulo. 484 pp. University's Natural History Reservation (FNHR) following es- FERNANDEZ, F. A. S. 1995. Metodos para estimativa de parametros tablishment of the area as a reserve. Although protected from an- populacionais por captura-marcacao-recaptura. Oecologia Brasiliensis thropogenic alterations, the area has undergone continual and pro- 2:1-26. gressive change due to natural ecological succession (Fitch 1999; HEYER, W. R., A. S. RAND, C. A. G. CRUZ, AND 0. L. PEIXOTO. 1988. Deci- Fitch et al. 1984, 2001, 2003b). The closing in of forest and elimi- mations, extinctions, and colonizations of frog populations in South- nation of open places associated with agriculture and grazing has east Brazil and their evolutionary implications. Biotropica 20:230-235. been unfavorable for snakes. Several of the species have disap- HOULAHAN, J. E., C. S. FINDLAY, B. R. SCHMIDT, A. H. MEYER, AND S. L. peared from this 239-hectare tract, and almost all species have Kuzmusr. 2000. Quantitative evidence for global amphibian population declines. Nature 404:752-755. been drastically reduced (Fitch 1999). The adjoining Nelson En- MARTOF, B. S. 1953. Territoriality in the green frog, Rana clamitans. Ecol- vironmental Study Area (NESA), acquired by the University in ogy 34:165-174. 1970, has provided a contrast to FNHR in terms of habitat stabil- NASCIMENTO, D. 2003. Comportamento reprodutivo de Scinax albicans ity. Invasion of woody vegetation is prevented on NESA by the (Bokermann, 1967) (Amphibia, Anura, Hylidae), na floresta pluvial regular mowing of blocks of former pasture and cultivated land, montana no sudeste do Brasil. Unpubl. M.Sc. Dissertation, Museu and as a result, existing snake populations resemble those found Nacional, Rio de Janeiro. on FNHR in earlier stages of succession (Fitch 1999). PECHMANN, J. H. K., D. E. SCOTT, R. D. SEMLITSCH, J. P. CALDWELL, L. J. Sampling areas (House Field, Quarry Field, etc.) are described Vrrr, AND J. W. GIBBONS. 1991. Declining amphibian populations: the by Fitch (1999). Of the 12 censuses reported here, eight were from problem of separating human impacts from natural fluctuations. Science 253:892-895. FNHR and were done in the earlier years of study (before 1979) SEBER, G. A. F. 1982. The Estimation of Abundance and Related except that for Thamnophis sirtalis. Of the remaining four spe- Parameters. 2nd ed. Charles Griffin, London. 654 pp. cies, Carphophis vermis was censused by Clark (1970) on private WEYGOLDT, P. 1989. Changes in the composition of mountain stream frog property adjoining FNHR and three, Lampropeltis triangulum, L. communities in the Atlantic Mountains of Brazil: frogs as indicators of calligaster, and Coluber constrictor, were censused post-1990 on environmental deteriorations? Stud. Neotrop. Fauna Environ. 243:249- NESA. 255. Unless otherwise noted, density estimates are based on field YOUNG, B. E., K. R. LIPS, J. K. REASER, R. IBAREZ, A. W. SALAS, J. R. records and censuses originally published in Fitch (1999) except CEDERO, L. A. COLOMA, S. RON, E. LA MARCA, J. R. MEYER, A. MuSroz, for Clark's (1970) Carphophis figures. Mark-recapture records and F. BOLAS1OS, G. CHAVES, AND D. Rollo. 2001. Population declines and priorities for amphibian conservation in Latin America. Cons. Biol. the Petersen Index were the bases for the abundance estimates for 15:1213-1223. most species, including Agkistrodon contortrix (Copperhead), ZAR, J. H. 1999. Biostatistical Analysis. 4th ed. Prentice Hall, Englewood Carphophis vermis (Western Wormsnake), Coluber constrictor Cliffs, New Jersey. 663 pp. (Racer), Crotalus horridus (Timber Rattlesnake), Diadophis

Herpetological Review 37(2), 2006 161 punctatus (Ring-necked Snake), Nerodia sipedon (Northern of C. horridus from FNHR was in 1964. By contrast, 12 were Watersnake), Pantherophis obsoletus (Black Ratsnake), Pituophis captured from a 10-ha area of NESA (Biotic Succession Area) in catenifer (Bullsnake), and Thamnophis sirtalis (Common the years 1990 through 2002 (Fitch et al. 2003). Additionally, in Gartersnake). For the remaining three species, Lampropeltis the spring of 2003, 26 were captured along a rock outcrop about calligaster (Prairie Kingsnake), Lampropeltis triangulum 100 m long near Frank B. Cross Reservoir, in an area adjoining (Milksnake), and Storeria dekayi (DeKay's Brownsnake), densi- NESA on the east (Fitch et al. 2004). ties were estimated from their respective numbers relative to one Diadophis punctatus-Many different censuses are available or more species with larger mark-recapture samples. for the once-abundant Ring-necked Snake (Fitch 1999). Seven were Weights are based on figures from Fitch (1999, Table 98). Num- done in Quarry Field in 1966-67 and 1969-70, with sampling pe- ber of snakes, weight, and biomass per hectare were log trans- riods ranging from 0.5 to 2 months Eliminating the extreme low- formed for the regressions. Representative weight for each spe- est and highest estimates (597 and 4000/ha) gave a range of 791- cies is the midpoint between the means for the adults of both sexes 2039/ha and a mean of 1325/ha. An additional eight censuses were (excluding gravid and recently parturient females). For the sake from House Field with sampling periods ranging from 0.5 to 3 of consistency, only adult mass was used to calculate biomass from months (1965-1967); excluding the lowest and highest estimates density, although immatures were present for every species (262 and 1792) gave a mean of 1224/ha (range = 773-1761). Av- censused (see Fitch 2000, for age pyramids for 10 of the species). eraging the means for these two areas, both on FNHR, gives an Attempting to factor in mean mass across all sizes had the poten- overall mean of 1275/ha. tial of introducing more bias than was inherent in excluding these Lampropeltis calligaster-2.5/ha, 1990-1997, on the northwest- other size classes. Relative abundance of life history stages varied ern pens area of NESA. This estimate is the average of 1.6/ha markedly over each trapping season, but these seasonal fluctua- (based on ratio of capture records compared to Thamnophis) and tions were not synchronized among species. Also, age composi- 3.3/ha (compared to Coluber). Since 2001, 14 L. calligaster have tion of each species censused was partly a function of trapping been captured on NESA, and only two individuals (both in 2002) method. For example, entrance holes of the wire funnel traps were have been found on FNHR. mostly too small to allow entry of large adult Timber Rattlesnakes. Lampropeltis triangulum-0.6/ha, 1990-1997, on the north- In contrast, the traps were well suited to catch larger young and western pens area of NESA. This estimate is the average of 0.52/ adults of the Common Gartersnake, while tiny neonates of this ha (compared to Thamnophis) and 0.73/ha (compared to Coluber). species easily passed through the quarter-inch mesh of the traps, The last capture of L. triangulum on FNHR was in 1999. Since and hence are missing from the trapping records. More informa- 2001, 17 have been captured on NESA. tion is available in Fitch (1999), including details of food habits Nerodia sipedon-The majority of records were concentrated and population fluctuations. Age structure pyramids and "stand- about the FNHR pond, but a few were scattered over a much larger ing crop" biomass estimates for 10 local snake species are pre- area, reflecting the fact that these snakes travel, following the water sented in Fitch (2000). supply in drainage systems. The Petersen Index indicated 10 snakes for 1978. The area covered was approximately 25 ha, resulting in RESULTS an estimate of 0.4/ha (previously unpublished). Dates of census, sampling area, abundance, mean adult weights, Pantherophis obsoletus-Records for the Black Ratsnake are and other numbers based on these figures are listed in Table 1. perhaps not sufficiently concentrated in time to yield a reliable The following list explains the specifics of the abundance esti- sample free from the effect of temporal change, although survi- mate for each species. vorship in this species is longer than in most. Hence the Petersen Agkistrodon contortrix-18.6/ha is the mean of ten censuses in Index estimate may have some validity, despite the long sampling 1977 on FNHR, five for House Field and five for Quarry Field. period (1950 through 1960) and the large area involved (129.5 Carphophis vermis-729Iha in 1966 on private property 1 km ha), approximately the northern half of the Reservation. Based on west of FNHR headquarters (Clark 1970). This was the highest varying sampling and resampling times, densities of 0.9 to 3.6/ha density estimate given by Clark (1970). We use it because it was have been calculated (Fitch 1999, Table 101). The average of these. derived with the Petersen Index, as were the estimates for the other 2.3/ha, is used for this study. species reported here. Clark obtained a lower estimate with the Pituophis catenifer-From the start, the Bullsnake was unfa- Hayne modification of the Lincoln Index, but this modification is vorably affected by successional changes, and it dwindled through apparently more appropriate for small mammals than for snakes the 1950s and 1960s (Fitch 2003). Censuses, taken almost entirely (Carpenter 1952). from the northwest quarter-section (64.7 ha) of FNHR in 1958, Coluber constrictor-13.2/ha based on five separate 0.5-month 1959, 1963, 1964, and 1966 averaged 38.5 snakes per year, giving sampling periods in 1991-1994 on the northwestern pens area of an estimate (previously unpublished) of 0.60 snakes/ha. The last NESA. These five were chosen from a number of available cen- FNHR capture of this species was in 1984. Six were captured on suses for this species (Fitch 1999) because all included more than NESA during the period 1997-2002. one recapture, and shorter sampling periods generally seem to yield Storeria dekayi-Estimated at 42/ha in the House Field-Quarry the most reliable estimates for this species. Field area, 1966-67 and 1969-70, based on a 3.3% ratio of brown Crotalus horridus-0.2/ha for the northwestern quarter of snakes to the more readily censused Diadophis. FNHR in the early 1960s from a Petersen Index estimate (previ- Thamnophis sirtalis-15.4/ha in House Field, 1988-1997. Al- ously unpublished) based on only two recaptured rattlesnakes, at though the Common Gartersnake was abundant enough to pro- a time when the species was already dwindling. The last capture vide a surfeit of samples from several areas, only samples from

162 Herpetological Review 37(2), 2006 TABLE 1. Abundance and biomass of 12 snake species on the Fitch Natural History Reservation (FNHR), a plot of private land adjoining FNHR (Carphophis), and Nelson Environmental Study Area (NESA).

Species Site of Year(s) of Number Percentage of total Mean adult Biomass per Percentage of census census individuals/ha number of snakes weight (g) hectare (g/ha) total snake biomass/ha

Agkistrodon contortrix FNHR 1977 18.6 0.89 150 2790 13.4 Carphophis vermis 1 km W FNHR 1966 729.0 34.72 7 5103 24.5 Coluber constrictor NESA 1991-1994 13.2 0.63 140 1848 8.9 Crotalus horridus FNHR 1960-1963 0.2 0.01 713 143 0.7 Diadophis punctatus FNHR 1965-67 1275.0 60.72 6 7650 36.7 1969-70 Lampropeltis calligaster NESA 1990-1997 2.5 0.12 202 505 2.4 Lampropeltis triangulum NESA 1990-1997 0.6 0.03 73 44 0.2 Nerodia sipedon FNHR 1978 0.4 0.02 202 81 0.4 Pantherophis obsoletus FNHR 1950's 2.3 0.11 392 902 4.3 Pituophis catenifer FNHR 1958-1966 0.6 0.03 671 403 1.9 Storeria dekayi FNHR 1966-67 42.0 2.00 5 210 1.0 1969-70 Thamnophis sirtalis FNHR 1988-1997 15.4 0.73 77 1186 5.7

House Field (10 ha area, FNHR) are reported here. In this area, shrub/forest encroachment (Fitch et al. 2001). The three censuses snakes were found beneath shelters, and the hand captures included on NESA were done between 1990-1997; all three were at the first-year young that can escape through quarter-inch mesh of live same site, one that resembled earlier stages of succession on FNHR. traps used, for example, at the NESA site. Of 44 census figures For these reasons, we discuss these figures as if they were ac- available from 1985-1997 (Fitch 1999), 13 were selected because quired from the same area in the same years. Our premise is that each included more than one recapture and each was conducted in the numbers reported here are representative of relative snake abun- approximately two consecutive months of sampling (first month dance during the earlier stages of succession on FNHR. for primary sample followed by a 3-4 week re-sampling period). Among the 12 species of FNHR/NESA snakes yielding suffi- Shorter and consecutive sampling periods are probably more reli- cient data (Table 1, Fig.1), there was a statistically significant in- able for this species because longer sampling periods may result verse relationship (r = - 0.85, P < 0.001) between typical adult in inflated estimates (Fitch 1999). weight of each species and number of snakes/ha (Fig. 1), imply- ing that smaller snakes tend to be more abundant than larger spe- DISCUSSION cies. There was also a statistically significant (r = 0.82, P = 0.001) The mark-recapture method of assessing population size is, in relationship between number of individuals/ha and biomass/ha, most cases, the most available method for assessing the numbers suggesting that the more abundant species tend to form a larger of a reasonably abundant species. However, numbers acquired portion of the snake biomass per unit area. through this approach are perhaps most appropriately compared Consistent with the inverse relationship between size of snake for similarities/differences in orders of magnitude (Fitch 1975). A and number/ha, the highest population numbers were recorded for further caveat is that not all censuses were completed in exactly the three species of smallest snakes. These were also the only spe- the same area and time for each of the 12 species. However, most cies specializing in a diet of earthworms and/or slugs (Fitch 1999). of those from FNHR were done in the same or overlapping areas Diadophis punctatus outnumbered (60% of total numbers) the of the Reservation before ecological succession had resulted in population estimates for all other species combined. Its numbers

Herpetological Review 37(2), 2006 163 4 ways. Pituophis, Crotalus, and Lampropeltis triangulum were among the first to disappear from FNHR as succession progressed,

) Dpun r = - 0.85, P <0.001 and dense undergrowth and unbroken forest canopy invaded pre- 3 • Cver

log,o viously open fields and forest-edge habitat. Although the rattle-

( snake is a forest inhabitant, it apparently requires open sunny places ha / 2 for basking. Local Milksnakes prefer open woodland or forest edge Sdek kes e with flat rocks, and the Bullsnake requires short-grass habitat. •Acon • Tsir Pocket gophers (Geomys bursarius) and Five-lined

f sna • Ccon (Eumeces fasciatus), favored prey for, respectively, Bullsnakes and o Lcal Pobs Milksnakes, also disappeared or became scarce in the early years ber •

m of FNHR succession (Fitch 1999; Kettle, Fitch, and Pittman, 0 Uri •Pcat Nu * •Nsip unpubl. ms.). • Chor Despite relatively low abundances (< 3/ha), Lampropeltis

calligaster and Pantherophis obsoletus, particularly the latter be- 05 1 0 1 5 2.0 2.5 3.0 cause of its greater bulk, constituted a moderate proportion of the Weight in grams (log 10) total biomass represented by the nine larger snake species. Our figure of 2.3/ha for the Black Ratsnake is comparable to the den- Flo. 1. Relationship between a typical adult weight of a snake species sity (3.9-4.2/ha) reported by Weatherhead and Haysack (1989) and number per unit area. Species codes (in parentheses) are as follows: for a population at the northern edge of the species' range in east- Agkistrodon contortrix (Acon), Carphophis vermis (Cver), Coluber con- ern Ontario. Of the less abundant forms so far discussed, the Black strictor (Ccon), Crotalus horridus (Chor), Diadophis punctatus (Dpun), Ratsnake is the only species that seemed to maintain a relatively Lampropeltis calligaster (Pcal), Lampropeltis triangulum (Ltri), Nerodia sipedon (Nsip), Pantherophis obsoletus (Pobs), Pituophis catenifer (Pcat), stable population despite the successional changes on FNHR. The Storeria dekayi (Sdek), and Thamnophis sirtalis (Tsir). Prairie Kingsnake, by contrast, disappeared from FNHR as en- croachment of brush and forest replaced its preferred tallgrass habitat. (mean 1275/ha) are comparable to those (1289/ha, censused by The three remaining species, the Copperhead, Racer, and Com- quadrat counts) reported by Godley (1980) for the Striped Swamp mon Gartersnake, were all prominent members of the local snake Snake, Regina alleni, in two water hyacinth-choked canals in south- fauna. Because they were present in approximately comparable ern Florida. To our knowledge, these figures are the highest known numbers, their relative contributions to the overall snake biomass for any North American snake species, excluding reproductive and were determined more by weight than by large differences in abun- denning aggregations. Godley (1980) stated that his estimated mean dance. The average Copperhead weighed about twice as much as standing crop for the Regina population (30.8 kg/ha) was greater a Common Gartersnake and slightly more than the Racer, thus than that known for any snake species; his estimate far exceeds Copperheads constituted the most substantial portion (35%) of the our highest (7.65 kg/ha for Diadophis). In a study of another di- biomass of the nine larger species, followed by the Racer (23%) minutive species, Clark and Fleet (1976) reported a range of 229 and the Common Gartersnake (15%). Hirth and King (1968) re- to 348/ha (mark/recapture, Lincoln Index) for the Rough ported a mid-summer density of 49g/ha for Coluber constrictor Earthsnake, Virginia striatula. Like Diadophis in northeast Kan- mormon, in desert habitat in northwestern Utah, a figure that seems sas, Virginia in eastern Texas apparently specializes in a diet of strikingly low when compared with our figure of 1848g/ha for C. earthworms (Clark and Fleet 1976). c. flavivientris in northeastern Kansas. With the caution that fall The second and third most abundant species were Carphophis samples "...may be subject to minimum bias compared with and Storeria. Excluding Diadophis, wormsnakes was more abun- samples collected at other times of year...", Fitch (2000) reported dant (729/ha) than all remaining species combined, and accounted a much lower density (4.9/ha) and mean weight (103.6g) for fall for 35% of the total snakes (24% of total snake biomass). The samples of Copperheads than we report here. This difference re- diminutive natricine, Storeria dekayi (42/ha), was several times emphasizes the fact that local populations show large fluctuations more abundant than an estimate (14/ha) based on mark-recapture in abundance and that census figures are rough approximations for the same species in Ontario, Canada (Freedman and Catling best used for relative comparisons and assessed in terms of orders 1978), but it should be noted that our estimate was indirect, based of magnitude difference. on a ratio to the more adequately sampled Diadophis. Our estimate of 15.4/ha for Thamnophis sirtalis is well within Among the remaining nine species, primary prey consisted of the range of values reported for this species: 1.7 in British Colum- vertebrates except for first-year garter snakes that tended to feed bia (Farr 1988 as cited by Rossman et al. 1996) and 89 in Ohio predominantly on earthworms (Fitch 1999). Among these nine (Reichenbach and Dalrymple 1986). The latter study estimated a species, four (Timber Rattlesnake, Northern Watersnake, biomass density of 2.8-5.5 kg/ha for T. sirtalis in Ohio, 2.3-4.6 Milksnake, Bullsnake) occurred at densities of less than one per times greater than our estimate for Kansas garter snakes. Among hectare. Even at such low densities, the Timber Rattlesnake con- those snake species that have been abundant on FNHR, the Com- tributed 3-4 times the biomass/ha of that estimated by Hirth and mon Gartersnake has perhaps been the most persistent, and was King (1968) for a congener, the Great Basin Rattlesnake, C. one of the few species of this study that maintained sufficiently ore ganus lutosus, in desert habitat of northwestern Utah. Nerodia high population numbers to produce a useful FNHR estimate in was fairly localized around the FNHR pond and associated water- the 1990's. Among the 12 snakes we studied, this species might

164 Herpetological Review 37(2), 2006 be considered the most generalized in food habits, as it consumes sas Biotic Succession Area. J. Kansas Herpetol. 8:20-21. invertebrates, amphibians, mammals, and birds. Although found , P. VON ACHEN, AND A. F. ECHELLE. 2001. A half century of forest in field habitats, it is also relatively tolerant of shade, and, as stated invasion on a natural area in northeastern Kansas. Trans. Kansas Acad. by Fitch (1999, Table 102), its population fluctuations seem to be Sci. 104:1-17. AND G. L. PITMAN. 2003. Probable succession related more affected by annual weather changes than by succession. Such prey changes of long-eared owls in Kansas. Kansas Onitholog. Soc. plasticity helps explain the wide geographic range and high abun- Bull. 54:42-43. dances of this species. FREEDMAN, W., AND P. M. CATLING.1978. Population size and structure of Parker and Plummer (1987) and Iverson (1982) provide gen- four sympatric species of snakes at Amherstburg, Ontario. Can. Field- eral reviews of snake densities, and Ernst and Barbour (1989) and Nat. 92:167-173. Rossman et al. (1996) summarize the data for various species of GODLEY, J. S. 1980. Foraging ecology of the striped swamp snake, Regina Thamnophis. A comparison of the numbers reveals extensive in- alleni, in southern Florida. Ecol. Monog. 50:411-436. ter- and infra-specific variation in density patterns. As documented HIRTH, H. F., AND F. W. KING. 1968. Biomass densities of snakes in the for snakes in the vicinity of FNHR, the extent of intraspecific fluc- cold desert of Utah. Herpetologica 24:333-335. IVERSON, J. B. 1982. Biomass in turtle populations: a neglected subject. tuations in a single area can rival that attributable to geographic or Oecologica 55:69-76. interspecific variation (Fitch 1999). PARKER, W. S., AND M. V. PLUMMER. 1987. Population ecology. In R. A. Seigel, J. T. Collins, and S. S. Novak (eds.), Snakes: Ecology and Evo- Acknowledgments.-We thank those who, as graduate students, con- lutionary Biology, pp. 253-301. Macmillan Publishing Co., New York. tributed with their own studies of snake populations on or near the Reser- REICHENBACH, N. G., AND G. H. DALRYMPLE. 1986. Energy use, life histo- vation and have since maintained enthusiasm in this endeavor as is evi- ries, and the evaluation of potential competition in two species of gar- denced by their publications, including D. R. Clark, R. R. Fleet, W. S. ter snake. J. Herpetol. 20:133-153. Parker, G. R. Pisani, D. W. Platt, M. V. Plummer, and R. A. Seigel. Thanks ROSSMAN, D. A., N. B. FORD, AND R. A. SEIGEL. 1996. The Garter Snakes: also to S. F. Fox, J. F. Husak, and W. D. Kettle for their helpful discus- Evolution and Ecology. Univ. of Oklahoma Press. Norman, Oklahoma. sions, and to A. A. Echelle, R. N. Reed and three anonymous reviewers 332 pp. for advice and critical reading of the manuscript Finally, thanks are due WEATHERHEAD, P.J., AND D. J. HAYSACK.1989. Spatial and activity patterns to V. R. Fitch who helped with many aspects of the work reported in this of black rat snakes (Elaphe obsoleta) from radiotelemetry and recap- paper. ture data. Can. J. Zool. 67:463-468. WINNE, C. T., M. E. DORCAS, AND S. M. POPPY. 2005. Population struc- LITERATURE CITED ture, body size, and seasonal activity of black swamp snakes (Seminatrix pygaea). Southeast. Nat. 4:1-14. BONNET, X., D. PEARSON, M. LADYMAN, 0. LOURDAIS, AND D. BRADSHAW. 2002. `Heaven' for serpents? A mark-recapture study of tiger snakes (Notechis scutatus) on Camac Island, Western Australia. Austral Ecol. 27:442-450. Herpetological Review, 2006,37(2), 165-166. 0 2006 by Society for the Study of Amphibians and Reptiles CARPENTER, C. C.1952. Comparative ecology of the common garter snake (Thamnophis s. sirtalis), the ribbon snake (Thamnophis s. sauritus) The Tadpole of the Mexican Treefrog and Butler's garter snake (Thamnophis butleri) in mixed populations. Ecol. Monog. 22:235-258. Plectrohyla hazelae Taylor, 1940 CLARK, D. R., JR. 1970. Ecological study of the worm snake Carphophis vermis Kennicott. Univ. Kansas Publ. Mus. Nat. Hist. 19:85-194. MOISES KAPLAN , AND R. R. FLEET. 1976. The rough earth snake (Virginia striatula): Division of Reptiles and Amphibians, Museum of Zoology ecology of a Texas population. Southwest. Nat. 20:467-478. University of Michigan, Ann Arbor, Michigan 48109, USA ERNST, C. H., AND R. W. BARBouR.1989. Snakes of Eastern North America. e-mail: [email protected] George Mason Univ. Press, Fairfax, Virginia. 282 pp. FARR, D. R. 1988. The ecology of garter snakes, Thamnophis sirtalis and PETER HEIMES T elegans, in southeastern British Columbia. Master's Thesis, Univ. of 5114 Edgewood Place, Los Angeles, California 90019, USA Victoria, British Columbia. and (Diadophis Frrcu, H. S. 1975. A demographic study of the ringneck snake RAFAEL AGUILAR punctatus) in Kansas. Univ. Kansas Mus. Nat. Hist. Misc. Pub. 62:1- Manantiales 14, Barrio Santo Domingo 53. Teportlan, Morelos, Mexico . 1999. A Kansas Snake Community: Composition and Changes over 50 Years. Krieger Publ. Co. Inc., Malabar, Florida. 165 pp. Recently obtained tadpoles from the vicinity of Ixtlan de Juarez, . 2000. Population structure and biomass of some common snakes Sierra Juarez, Oaxaca, Mexico, were raised in captivity and deter- in central North America. Univ. Kansas Nat. Hist. Mus. Sci. Pap. 17:1- mined to be those of Plectrohyla hazelae. Herein, we describe the 7. tadpole of P hazelae. . 2003. Reproduction in snakes of the Fitch Natural History Res- ervation in northeastern Kansas. J. Kansas Herpetol. 6:21-24. All the tadpoles (UMMZ 236001-02) were collected on 7 May 2004, on muddy bottoms of shallow pools in one small creek 3.8 , V. R. FITCH, AND W. D. KETTLE. 1984. Reproduction, population changes and interactions of small mammals on a natural area in north- km from "Rancho Tejas," Sierra de Juarez, on the Ixtlan de Juarez- eastern Kansas. Univ. Kansas Mus. Nat. Hist. Occ. Pap. 109:1-37. Rancho Tejas road (17°18'96", 96°26'61"; 1900 m elev.). The tad- , G. R. PISANI, H. W. GREEN, A. F. ECHELLE, AND M. ZERWEKH. 2004. poles were taken to Mexico City (2400 m) and raised to adult A field study of the timber rattlesnake in Leavenworth County, Kan- stage except for few which were killed in 10% formalin at stages sas. J. Kansas Herpetol. 11:18-24. 30, 34, 36, 38, 39, 42, and 43 (Gosner 1960).Terminology and , S. SHARP AND K. SHARP. 2003. Snakes of the University of Kan-

Herpetological Review 37(2), 2006 165 processes; lower jaw sheath moderately keratinized, V-shaped, bearing 20-25 small round serrations. In life, body dark gray with slightly darker blotches. In preser- vative, body gray brown, tail musculature gray beige, and blotches dark brown; large dark blotches scattered on dorsal and lateral parts of body, anterior surface of oral disc, dorsum of legs, tail 1 cm musculature and fins; fins more pigmented on posterior 2/3 of tail than on anterior 1/3; tail musculature more pigmented on anterior than posterior half of tail; rims of vent, nostrils, and spiracle darkly pigmented. Keratin can be present or absent on both jaw sheaths, or present only on the lower jaw. The keratin on the lower jaw can form a narrow continuous arch or two discontinuous patches. The mar- ginal papillae can be well or poorly defined on the anterior edge of the oral disc. In dorsal view, the body is ovoid (Stages 36 and younger) or elongated (Stages 37 and older). The tadpoles of Plectrohyla hazelae differ from those of other stream-dwelling species of Plectrohyla from the Sierra de Juarez (i.e., P. bistincta, P. crassa, P. calthula, P. celata, P. cembra, P. 110fieffhwu —....11thilligii1110110 cyanomma) by having a tail with its highest point at the posterior 2/3 and by lacking one or several rows of large submarginal papil- lae anterior to A-1 and/or posterior to P-3.

Acknowledgments.—We thank John Megahan for helping with fig- 1 mm ures and Oscar Flores for collecting permits.

LITERATURE CITED FIG. 1. Tadpole and oral disc of Hyla hazelae (Stage 38) (UMMZ). ALTIG, R., AND R. W. MCDIARMID. 1999. Body plan: development and morphology. In R. W. McDiarmid and R. Altig (eds.), Tadpoles: The measurements follow those proposed by Altig and McDiarmid Biology of Anuran Larvae, pp. 24-51, Univ. Chicago Press, Chicago, (1999). Acronyms as follows: UMMZ = University of Michigan Illinois. Museum of Zoology. DUELLMAN, W. E. 2001. The Hylid Frogs of Middle America. Society for A typical tadpole in Stage 38 (Gosner 1960) (UMMZ 236001; the Study of Amphibians and Reptiles, Ithaca, New York. Fig. 1; measurements in mm): body length 15.6; tail length 29.7; GOSNER, K. L. 1960. A simplified table for staging anuran embryos and total length 45.3; body slightly depressed; snout round in dorsal larvae with notes on identification. Herpetologica 16:183-190. view and profile; eyes large (eye diameter 2.6), dorsolateral, bulgy, separated by a distance of 3.1; nostrils separated by a distance of 3.4, slightly closer to eyes than to tip of snout; spiracle sinistral, C-type (Fig. 3.1 in Altig and McDiarmid 1999), directed posterodorsally, angled 80° with respect to longitudinal axis of body, opening below midline of body and half way between tip of snout and posterior end of body; vent tube dextral, G-type (Fig. 3.5 in Altig and McDiarmid 1999); width of caudal musculature 5.1; dorsal fin extending slightly onto body; maximum height of tail located at posterior 3/4 of tail; tip of tail round (partly dam- aged in this individual). Oral disc medium size (oral disc diameter/max. body width = 0.5), elliptical, ventrally located, not emarginate, bordered by marginal papillae laterally and posteriorly; marginal papillae on lateral and posterolateral parts of oral disc biserial; marginal pa- pillae of posterior part of oral disk slightly offset, intercalated, biserial to triserial; 4 to 5 large submarginal papillae on each side of oral disc; labial tooth row formula 2(2)/3; gap of A-2 1/5 of row length; lengths of A-1 and P-2 equal, reaching submarginal pa- pilla; relative lengths of posterior teeth rows P-2>P-1>P-3; P-3 distinctively shorter than other rows; labial teeth of P-3 shorter than those of other rows; labial tooth ridges rigid; upper jaw sheaths Juvenile female ceratophora: Fort Worth Zoo specimen. Digi- poorly keratinized, bearing small round serrations but not lateral tal photo illustration by Clay M. Garrett.

166 Herpetological Review 37(2), 2006 area with a complex geologic history. Over the last 40 million Herpetological Review, 2006, 37(2), 167-170. 0 2006 by Society for the Study of Amphibians and Reptiles years interplate impact resulted in considerable uplift and volcan- ism and importantly the accretion of at least 32 tectonostratigraphic Checklist and Comments on the Terrestrial terranes along the northern leading edge of the island that have Fauna of Kau Wildlife Area, Papua New influenced the biodiversity of the region (Pigram and Davies 1987; Guinea Polhemus and Polhemus 1998). To date there has been no comprehensive herpetofaunal reports from the KWA. Here I compile a list of terrestrial reptile species CHRISTOPHER C. AUSTIN present in the KWA based on fieldwork over the past 14 years. Museum of Natural Science, 119 Foster Hall, Louisiana State University Baton Rouge, Louisiana 70803, USA The KWA terrestrial reptile fauna, exclusive of crocodylians and e-mail: [email protected] turtles, currently includes 25 and 7 snakes representing 8 families and 21 genera (Table 1). A similar compilation is under- The island of New Guinea has been identified as a megadiverse way for the amphibians of the region (S. Richards, pers. comm.). region because of its extraordinary biodiversity and highly en- Specific specimen and locality information as well as associated demic biota (Mittermeier and Mittermeier 1997). New Guinea, tissues can be accessed via a searchable database of the LSU Mu- the world's largest and highest tropical island, occupies less than seum of Natural Science reptile and amphibian collection (http:// 1% of global land area yet 5-7% of the world's biodiversity is www.lsu.edu/museum). found on the island (Beehler 1993; Dinerstein and Wikramanayake 1993; Mack 1998; Myers et al. 2000). The herpetofauna of New SPECIES RICHNESS, AND SPECIES-COMPLEX GROUPS Guinea currently known to science accounts for about 5% of the There are a large number of species complexes in the diverse world's reptile and amphibian diversity (Allison et al. 1998). Re- New Guinea reptile fauna, especially among the scincid lizards. markably, this is an underestimate of true diversity; it is predicted In addition to species complexes, many currently recognized spe- that 30-60% of the reptile and amphibian fauna of New Guinea cies likely represent more than one taxon. Species richness, there- remains unknown to science (Allison 1993). The vast diversity of fore, is likely much greater than is currently recognized. Below I biological life on New Guinea is a result of the island's diverse comment on and address some of the taxonomic impediments to topography, extensive range of habitat types, and complex geo- understanding the KWA terrestrial reptile fauna. logical history. With elevations ranging from sea level to over 5000 The agamid genus Hypsilurus is a poorly understood group. The m, the varied habitat zones, packed into an area one-tenth the size taxonomy of the genus has been muddled by inadequate original of the United States, include relictual tropical glaciers, alpine grass- descriptions, misidentified museum specimens, and the fact that lands, montane moss forests, dense lowland rainforests, sago palm the last comprehensive work on this group is 90 years old (de swamps, and eucalypt savannas. Rooij 1915). In particular, the geographic distribution and spe- The lowland rainforest on the north coast of Papua New Guinea cific-level variation for virtually all species of Hypsilurus is not has been severely impacted by logging (Beehler 1993). One of the well understood. A recent comprehensive review of all type mate- few areas of lowland rainforest set aside for conservation and sci- rial has provided a much needed taxonomic summary and clarifi- entific study is the Kau Wildlife Area (KWA: 05°09'S, 145°46'E) cation (Manthey and Denzer 2006). Manthey and Denzer recog- near the provincial capital of Madang, Madang Province. The KWA nize 14 species of Hypsilurus acknowledging that this diversity is is community owned and managed by the Didipa Clan of Kau and undoubtedly an underestimate (Manthey and Denzer 2006; Moody Baitabag Villages. The 800-ha KWA reserve is part of the exten- 1980). There are two species of Hypsilurus in the KWA: H. sive and broadly continuous Northern New Guinea lowland modestus and H. papuensis. Hypsilurus modestus is a relatively ecoregion that is made up of lowland, freshwater, and peat swamp common small-bodied (maximum SVL = 107 mm) Hypsilurus forests. The lowland forests and freshwater swamps from this with a broad range throughout the Papuan region (New Guinea ecoregion contain diverse habitats, including lowland and hill for- and nearby associated islands), whereas H. papuensis is large bod- est, grass swamps, swamp forests, savannas, and woodlands (Conn ied (maximum SVL = 190 mm) and uncommon with a poorly 1995; Gressitt 1982; Henty 1981; Womersley 1978). The KWA, identified distribution throughout the Papuan region. between 20-70 m in elevation, includes primary, successional, and The gekkonid genus Nactus includes the widespread Nactus riparian lowland broadleaf-evergreen forest. As many as 155 spe- pelagicus complex that undoubtedly consists of several distinct cies of trees have been identified within a 1-ha plot of primary species, the identification of which has been hindered by morpho- forest in the KWA (Bonaccorso et al. 2002). Dense stands of sago logical conservatism and lack of adequate study. Based on mor- palm (Metroxylon sagu) are found in the low wet areas along the phological data, Zug and Moon (1995) determined the distribu- Kau and Biges Rivers. Many areas of the KWA have historically tion of the asexual N. pelagicus, which includes Micronesia, south- been subjected to traditional shifting fruit and vegetable gardens ern Vanuatu (Erromango and Tanna Islands), New Caledonia, and and many old abandoned garden areas surround the reserve. The eastward to Melanesia and Polynesia. Nactus multicarinatus, a climate of this region is wet tropical forest and in nearby Nagada bisexual species, has a range from the southern Solomon Islands Harbor annual precipitation averaged 3460 mm from 1994-96. and Vanuatu (excluding the islands of Tanna and Erromango) (Zug The area is typified by distinct wet and dry seasons with less than and Moon 1995). Donnellan and Moritz (1995) identified two 100 mm of monthly precipitation from June through August highly differentiated populations of the Nactus pelagicus com- (Bonaccorso et al. 2002). plex in Madang Province, Papua New Guinea based on allozymes. The northern region of New Guinea is a very active tectonic One of these Madang populations showed no fixed differences

Herpetological Review 37(2), 2006 167 TABLE 1. Checklist of the terrestrial reptile fauna of Kau Wildlife Area, Papua New Guinea. I refer to a species as 'Com- mon' if it is typically encountered in an appropriate 8-h search period. Species listed as 'Uncommon' are encountered infre- quently in the KWA and typically require more than a single day/night search of 8 h to locate. Species Comments & Literature

Lizards Agamidae Hypsilurus modestus Common (Manthey and Denzer 2006; Moody 1980) Hypsilurus papuensis Uncommon (Manthey and Denzer 2006; Moody 1980) Gekkonidae Nactus multicarinatus Common (Donnellan and Moritz 1995; Moritz 1987; Zug and Moon 1995) Cyrtodactylus sp. Uncommon (Brown and Parker 1973) Gekko vittatus Common (de Rooij 1915) Gehyra sp. Uncommon (Beckon 1992; Chrapliwy et al. 1961; King 1984; King and Homer 1989) Hemidactylus frenatus Common (Mortiz et al. 1993) Lepidodactylus lugubris Common (Mortiz et al. 1993) Scincidae Carlia mysi Common (Zug [2004] revised the Carlia fusca complex) caeruleocauda Common (Brown 1991) Emoia longicauda Uncommon (Brown 1991) Emoia jakati Common (Brown 1991) Emoia kordoana Uncommon (Brown 1991) Lamprolepis smaragdina Common (Greer 1970) Lipinia noctua Uncommon (Austin 1998; Zweifel 1979) Lobulia brongersmai Uncommon (Allison and Greer 1986; Zweifel 1972) Prasinohaema virens Uncommon (Mys 1988) Sphenomorphus jobiensis Common (Donnellan and Aplin 1989) Sphenomorphus mulleri Uncommon (de Rooij 1915) Sphenomorphus simus Common (formally S. stickli, Shea and Greer 1999) Sphenomorphus solomonis Common (de Rooij 1915) Sphenomorphus derooyae Uncommon (de Rooij 1915) Triblonotus gracilis Uncommon (Cogger 1972; Zweifel 1966) Varanidae Varanus indicus Common (low density) (Bohme 2003) Varanus prasinus Uncommon (low density) (Bohme 2003; Sprackland 1991) Snakes Boidae Candoia aspera Common, the most common terrestrial snake found in Kau (Austin 2000) Candoia carinata Uncommon (Austin 2000) Pythonidae Morelia viridis Uncommon (low density) (Rawlings and Donnellan 2003) Colubridae Boiga irregularis Uncommon (O'Shea 1996) Stegonotus modestus Uncommon (O'Shea 1996) Stegonotus parvus Uncommon (O'Shea 1996) Elapidae Micropechis ikaheka Uncommon (O'Shea 1996)

with the Solomon and northern Vanuatu bisexual Nactus tact zone, if there is one, between the two genetically distinct multicarinatus populations and thus this name should apply to one Madang populations has not been identified and it is possible that of the two Madang populations (Zug and Moon 1995). The con- both populations, likely corresponding to two distinct species, occur

168 Herpetological Review 37(2), 2006 in the KWA. recorded from Madang Province and likely occur in the KWA The gekkonid genus Gehyra in New Guinea consists of several (O'Shea 1996). (6) Acanthophis spp. have been recorded from species, the taxonomy of which is in need of revision (King 1984; Madang Province, but are typically found in open grassland not King and Horner 1989). The geographic distribution and specific- heavily forested regions like the KWA (O'Shea 1996). Various level variation for virtually all species of New Guinea Gehyra is specific epithets have been given to the several geographic races not well understood and many collections have misidentified taxa. of New Guinea populations of Acanthophis, yet authors differ in The scincid Sphenomorphus jobiensis complex was first identi- their assignment and the taxonomy of this group needs examina- fied from allozyme data and, to a limited extent, morphology, but tion (McDowell 1984; Storr 1981; Wiister et al. 2005). these data were not used to delineate species boundaries (Donnellan and Aplin 1989). DNA sequence data (Austin, unpubl. data) con- Acknowledgments.-I thank the people of KWA for the privilege to firm the apparent specific-level differentiation found by Donnellan conduct fieldwork on their land. I also thank I. Bigilale and F. Bonaccorso and Aplin (1989). In addition to the S. jobiensis complex, there from the PNG National Museum for their support of my field efforts. B. Roy, V. Kula, and B. Wilmot from the PNG Department of Environment are many other complexes that involve multiple cryptic species. and Conservation, and J. Robins from the PNG National Research Insti- These include the complexes within the genus Sphenomorphus tute provided assistance with research visas and export permits. This re- (S. derooyae, S. leptofasciatus, S. mulleri, S. pratti, and S. search was funded in part by the National Science Foundation (DEB solomonis, complexes). Sphenomorphus derooyae is likely a com- 0445213) and a Louisiana State University Faculty Research grant. I also plex of several species with many names available for different thank A. Bauer and L. Grismer for helpful comments on an earlier ver- populations (cranei, derooyae, maindroni, and wo/fi; G. Shea, pers. sion of this manuscript. comm.). In addition, other problematic groups include the Lipinia noctua complex (Austin 1999a,b), Papuascincus stanleyanus com- LITERATURE CITED plex, Emoia longicauda complex, and Carlia fusca complex (Zug ALLISON, A. 1993. Biodiversity and conservation of the fishes, amphib- 2004). Only the latter complex has been adequately studied with ians, and reptiles of Papua New Guinea. In B. M. Beehler (ed.), Papua 14 morphologically distinct species identified and taxonomically New Guinea conservation needs assessment. Vol. 2, pp. 157-225. The delineating in the New Guinea region (Zug 2004). Carlia mysi, Biodiversity Support Program, Washington, DC. with a broad range across the northeast coast of New Guinea and , D. BICKFORD, S. RICHARDS, AND G. A. TORR. 1998. Herpetofauna. In A. L. Mack (ed.), A Biological Assessment of the Lakekamu Basin, the Bismarck Archipelago, is the only Carlia species recorded from Papua New Guinea, pp. 58-62. RAP Working Papers 9, Conservation the KWA. International, Washington DC. UNCONFIRMED SPECIES THAT POSSIBLY ARE PRESENT IN THE KWA , AND A. E. GREER. 1986. Egg shells with pustulate surface struc- tures: basis for a new genus of New Guinea skinks (Lacertilia: Species not collected or visually confirmed, but likely present Scincidae). J. Herpetol. 20:116-123. in the KWA, include (1) members of the Cryptoblepharus boutonii AUSTIN, C. C. 1995. Molecular and morphological evolution in South complex, which includes up to 36 'forms' (Mertens 1931) many Pacific scincid lizards: morphological conservatism and phylogenetic relationships of Papuan Lipinia (Scincidae). Herpetologica 51:291-300. of which are likely distinct species. Although not seen in the KWA, . 1998. Phylogenetic relationships of Lipinia (Scincidae) from New forest populations of Cryptoblepharus are often more secretive Guinea based on DNA sequence variation from the mitochondria! 12 than coastal intertidal populations. 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170 Herpetological Review 37(2), 2006 and Lim (1967), Murphy and Voris (1994), Stuart et al. (2000), Herpetological Review, 2006, 37(2), 171 - 173. 2006 by Society for the Study of Amphibians and Reptiles Voris and Murphy (2002), and Voris et al. (2002). Two subspecies (H. b. buccata and H. buccata nigroventralis) have been described Forensic Implications of Dorsal Scale Row Counts (Deuve 1970), though few researchers appear to be aware of these on Puff-faced Water Snakes (Colubridae: designations. Homalopsinae: buccata) Although morphological descriptions of this snake are provided by many researchers, there is marked variability in published ranges for dorsal scale row counts (Table 1). This has the potential to lead BARRY W. BAKER to confusion in law enforcement efforts to monitor trade in this U.S. National Fish & Wildlife Forensics Laboratory 1490 East Main Street, Ashland, Oregon 97520-1310, USA species, and has implications for the concept of "scientific cer- e-mail: [email protected] tainty" (Bird 2001) in identifying this snake in a forensic context. Snake skins in the leather trade are often dyed, obscuring any origi- "The Dorsals. These scales are perhaps the most useful of all guides in nal body coloration or banding patterns. Identification, therefore, enabling us to differentiate between species and genera, but authors have may rely on scale shape, keeled vs. smooth scale morphology, and not availed themselves of their full value." [Wall 1902:338] dorsal scale row counts to determine if a leather item is consistent with the species it was declared to be. Wildlife, including live , parts, or products manufac- One of the most widely available texts to assist law enforce- tured from wildlife, must be properly declared to species of origin ment personnel in their identification of snake skins in the leather when imported into, or exported from, the United States. Forensic trade is Mahnert (1981). The entry for H. buccata in this volume scientists at the U.S. National Fish and Wildlife Forensics Labo- lists the number of dorsal scale rows in this species as 43-47, ratory (U.S. Fish & Wildlife Service) assist law enforcement agen- citing Taylor (1965), a reference well known and extensively used cies in identifying animals and animal products in cases where by herpetologists. In this example, a dyed snake skin with a dorsal suspected wildlife violations have occurred (Goddard and Espinoza scale row count of 35 at mid-body could be interpreted to be in- 2000). While the Laboratory does not initiate or lead such investi- consistent with H. buccata, given the fact that Taylor reports the gations, it provides forensic support in a broad range of areas in- range for this species as 43-47. However, further review of the cluding species identification (morphological and molecular), cause literature shows marked variability in published scale row counts of death examination, forensic chemistry, digital evidence analy- for this species (Table 1). sis (computer, audio, video and photography), fingerprint, fire- Taylor (1965) noted that "The number of scalerows on the body arm, and tool mark analysis, and crime scene investigation. is an important character and the number may vary at different Rigorous methods for identifying species in the wildlife trade points on the body." His description is somewhat confusing though, are critical to law enforcement efforts to enforce wildlife legisla- in that he stated "...number of scalerows variable, 37-47" (Taylor tion and international treaties (e.g., CITES), and to facilitate the 1965:921), while at the same time noting "Variation in scalerows legal trade in wildlife. Here I review published accounts of dorsal at the middle of the body is 43-47, the usual number being 45" scale row counts in the puffed-faced water snake (Homalopsis (Taylor 1965:922). Though not explicitly stated, Taylor's report buccata) and their forensic implications for identifying this spe- of 37-47 scale rows presumably refers to variability across the cies in the wildlife trade. While this species is not listed on CITES, entire body length of an individual. enforcement personnel are often called upon to identify this snake Even more confusing is Smith's (1943) description. In his gen- to facilitate the legal trade in wildlife. I show that published scale eral description of the monotypic genus Homalopsis, he describes row counts vary considerably and summarize these for wildlife the scales as "...in 39 to 47 rows" (Smith 1943:390). However, law enforcement purposes. his more detailed entry under the species name Homalopsis buccata The puffed-faced water snake is exploited extensively in the lists "Scales in 43-47, usually 45, rows" (Smith 1943:43-47). In wildlife trade, where its skin is used to manufacture leather prod- neither description did he note body location, a critical variable in ucts (Stuart et al. 2000). Zhou and Jiang (2004) reported that be- interpreting scale counts. tween 1991 and 2001 alone, 1,448,134 skins of this species were Gyi (1970), in his extensive and often cited revision of the sub- imported into China for the leather trade. Stuart (2004) reported family Homalopsinae, reported dorsal scale rows in H. buccata as numerous live specimens in reptile trade shops in Vietnam. Jenkins "37-41 at midbody." However, a closer review of his raw data H. buccata as the third most common reptile and Broad (1994) list (Table 12, p. 141) shows a range of 35-47 scale rows at midbody. skin imported into the U.S. during the years 1984-1990, consist- In his key to the genera of the Homalopsinae, he describes ing of 1,645,448 skins. Homalopsis with "dorsal scales in 39-47 rows" (Gyi 1970:61). This species is distributed through southeast Asia, including parts The origin of this significant discrepancy by Gyi in these three of Bangladesh, India, Nepal, Myanmar, Thailand, Cambodia, instances is not clear. Malaysia, Indonesia, Laos, Singapore, and Vietnam (Al-Murani The earliest report of scale row counts for H. buccata uncov- 1990; David and Vogel 1996). Various other common names have ered in my review is Gunther (1864), who described the scales as H. buccata, including the Masked Water Snake been applied to ranging from 37-47 rows. The two most recent references (Fuchs (Murphy et al. 1998), the Dog-faced Water Snake (Campden-Main and Fuchs 2003; Stuebing and Inger 1999) also produced the wid- 1984) [this name is more commonly applied to Cerberus rynchops], est ranges (Table 1). While the notation of Fuchs and Fuchs and the Asian False Water Boa (Franke and Telecky 2001). Re- (2003:229) (see Table 1) is not elaborated, the inference is that views of the general morphology, ecology, and phylogenetics of scales typically occur in 43-47 rows, but are known to range from H. buccata (in addition to those in Table 1) are provided by Berry

Herpetological Review 37(2), 2006 171 TABLE 1. Published ranges for dorsal scale row counts in Homalopsis buccata.

Citation Dorsal scale rows (range) Body location Geography'

Al-Murani 1990:128 39-43 Mid-body

Bergman 1951:514 37-47 (citing Rooy 1916) 43-47 (citing Smith 1943) 33-41 (citing Kopstein 1930) Gallbladder2 Indonesia 34-40 (p. 514) Gallbladder2

Bosch 1985:30 37-47 Sulawesi

Campden-Main 1984:82 43-47 Mid-body Vietnam

Cox 1991:198 43-47 ("usually 45") Mid-body Thailand

David and Vogel (1996:183) 37-47 Mid-body Sumatra

Deuve 1970:179-185 37-47 (H. b. buccata) 35-39 (H. buccata nigroventralis) Laos

Fuchs and Fuchs 2003:229 (37) 43-47 (49) Mid-body —

Gunther 1864:285 37-47 British India

Gyi 1970:61, 138, 141 39-47 (p. 61) 39-45 (p. 138) Anterior Myanmar, Thailand, 37-47 (p. 141) Anterior Malaya, Sumatra, Java 37-41 (p.138) Mid-body 35-47 (p. 141) Mid-body 27-31 (p. 138) Anterior to vent 23-33 (p. 141) Anterior to vent

Lim 1964:182 > 35 Mid-body Malaysia

Mahnert 1981 43-47 Thailand (by citing Taylor 1965)

Manthey and Grossmann 1997:307, 357 35-47 Southeast Asia

Marx and Rabb 1972:78-79 Range not reported, Mid-body through range span listed as 11

Rooij 1917:186-187 37-47 Indo-Australian Archipelago Saint Girons 1972: 48,110-114 43-47 Cambodia

Smith 1943:390-391 39-47 (p. 390) Myanmar, Sri Lanka, India 43-47 (p. 391) ("usually 45, rows") (p. 391)

Stuebing and Inger 1999:96 32-48 Mid-body Borneo

Taylor 1965:921-922 37-47 (p. 921) 43-47 (p. 922) Mid-body Thailand ("usual number being 45") (p. 922) Mid-body

Tweedie 1983:17, 103 37-47 Mid-body Malay Peninsula (including parts of Thailand, Malaysia and Singapore)

'Refers to geographic coverage of the text 2lnterpreted to mean mid-body

172 Herpetological Review 37(2), 2006 37-49 rows. This count of 49 scale rows is the highest of any mid- FUCHS, K., AND M. Fuchs. 2003. The Reptile Skin: A Key-Feature in the body dorsal rows reported for this species. The lowest count at Identification of Lizards and Snakes. Edition Chimaira, Frankfurt am mid-body is reported by Stuebing and Inger (1999), who list a Main. 407 pp. range of 32-48. Interestingly, they do not elaborate on the low GODDARD, K., AND E. ESPINOZA. 2000. Wildlife. In J. A. Siegel, P. J. Saukko, end of their range or compare it to previous accounts, which are and G. C. Knupfer (eds.), Encyclopedia of Forensic Sciences, pp. 1423- 1432. Academic Press, New York. generally higher. The combined ranges of Fuchs and Fuchs (2003) GUNTHER, A. C. L. G. 1864. The Reptiles of British India. Oxford & IBH and Stuebing and Inger (1999) result in a published dorsal scale Publishing Co, New Delhi, Bombay, Calcutta. 452 pp. row count in H. buccata of 32-49 scales at midbody. This range is Gri, K. K. 1970. A revision of colubrid snakes of the subfamily significantly different from most accounts in Table 1, especially Homalopsinae. Univ. Kansas Publ., Mus. Nat. Hist. 20:47-223. that reported by Mahnert (1981), a reference used by many of JENKINS, M., AND S. BROAD. 1994. International Trade in Reptile Skins: A those responsible for monitoring the wildlife trade. Based on this Review and Analysis of the Main Consumer Markets, 1983-91. TRAF- review, it appears that the entire range of dorsal scale counts of FIC International, Cambridge, U.K. 68 pp. 32-49 at mid-body should be considered consistent with H. buccata LIM, B. 1964. Notes on the elephant's trunk snake and the puff-faced by wildlife law enforcement personnel. water snake in Kuala Lumpur. Malay. Nat. J. 18:179-183. MAHNERT, V. 1981. Identification aid to entire snake skins. In CITES - This review reiterates that both accurate and precise morpho- Identification Manual Volume 5: Parts and Derivatives II. IUCN, Gland logical descriptions are critical not only to taxonomic research, Switzerland. but to forensic efforts and the conservation of species. It is likely MANTHEY, U., AND W. GROSSMANN. 1997. Amphibien & Reptilien that many herpetologists are unaware that their research, even basic Siidostasiens. Natur and Tier Verlag, Munster. 512 pp. descriptions, may be used in a forensic and legal context. In addi- MARX, H., AND G. B. RABB. 1972. Phyletic analysis of fifty characters of tion, enforcement personnel must be made aware that published advanced snakes. Fieldiana Zool. 63:1-321. morphological descriptions may refer only to a limited range of MURPHY, J. C., T. CHAN-ARD, D. R. KARNs, K. M. SANDRICK, AND H. K. variability within a species. It appears that Taylor's (1965) data VORIS. 1998. The water snakes of Lake Songkhla. Tigerpaper 25:1-4. , AND H. K. VoRis. 1994. A key to the homalopsine snakes. The have been used inappropriately by some as a diagnosis for the Snake 26:123-133. species from its entire range. The wide geographic range of R001.1, N. 1917. The Reptiles of the Indo-Australian Archipelago. II Homalopsis buccata and additional research has revealed further Ophidia. E.J. Brill, Leiden 334 pp. variability in dorsal scale rows in this species. Additional docu- SAINT GIRONS, H. 1972. Les serpentes du Cambodge. Mem. du Mus. Nat. mentation of scale count variability in H. buccata outside the range d'Historie Naturelle, Nouvelle Serie A, Zoologie 74:1-170. summarized here is also warranted, as are similar reviews of other SMITH, M. A. 1943. The Fauna of British India, Ceylon and Burma, In- snake species in the wildlife trade. cluding the Whole of the Indo-Chinese Sub-Region. Reptilia and Am- phibia. Vol. III (Serpentes). Taylor and Francis, London. 583 pp. STUART, B. L. 2004. The harvest and trade of reptiles at U Minh Thuong Acknowledgments. -I thank Rick Brooks (USFWS), who prompted this review, and B. Stuart, B. Weissgold, R.N. Reed, B. Yates, and P. Trail National Park, southern Viet Nam. TRAFFIC Bull. 20:25-34. for editorial comments. , J. SMITH, K. DAVEY, P. DIN, AND S. G. PLATT. 2000. Homalopsine watersnakes; the harvest and trade from Tonle Sap, Cambodia. TRAF- LITERATURE CITED FIC Bull. 18:115-124. STUEBING, R. B., AND R. E INGER. 1999. A Field Guide to the Snakes of AL-MURANI, A. S. 1990. Microdermatoglyphics and Phylogenetic Rela- Borneo. Natural History Publications, Borneo. 254 pp. tionships of Homalopsine Snakes. Unpublished Ph.D. dissertation, New TAYLOR, E. H. 1965. The serpents of Thailand and adjacent waters. Univ. York University. 145 pp. Kansas Sci. Bull. 45:609-1096. TWEEDIE, M. W. F. 1983. The Snakes of Malaya. Singapore National Print- BERRY, P. Y., AND G. S. Lim. 1967. The breeding pattern of the puff-faced water snake, Homalopsis buccata Boulenger. Copeia 1967:307-313. ers Ltd, Singapore. 167 pp. VORIS, H. K., M. E. ALFARO, D. R. KARNs, G. L. STARNES, E. THOMPSON, BERGMAN, R. A. M. 1951. The anatomy of Homalopsis buccata. Proc. K. Ned. Akad. Wet., Ser. C, Biol. Med. Sci. 54:511-524. AND J. C. MURPHY. 2002. Phylogenetic relationships of the Oriental- BIRD, S. J. 2001. Scientific certainty: research versus forensic perspec- Australian rear-fanged water snakes (Colubridae: Homalopsinae) based tives. J. Foren. Sci. 46:978-981. on mitochondrial DNA sequences. Copeia 2002:906-915. , AND J. C. MURPHY. 2002. The prey and predators of homalopsine BOSCH, H. A. J. IN DEN. 1985. Snakes of Sulawesi: Checklist, Key and Additional Biogeographical Remarks. Zoologische Verhandelingen No. snakes. J. Nat. Hist. 36:1621-1632. 217, Leiden. 50 pp. WALL, E 1902. Aids to the differentiation of snakes. J. Bombay Nat. Hist. Soc. 14:337-345. CAMPDEN-MAIN, S. M. 1984. A Field Guide to the Snakes of South Viet- nam. Smithsonian Institution. Reprint edition, Herpetological Search ZHOU, Z., AND Z. JIANG. 2004. International trade status and crisis for snake Service & Exchange, Lindenhurst, New York. 114 pp. species in China. Conserv. Biol. 18:1386-1394. Cox, M. J. 1991. The Snakes of Thailand and Their Husbandry. Krieger Publishing Co., Malabar, Florida. 526 pp. DAVID, P., AND G. VOGEL. 1996. The Snakes of Sumatra: An Annotated Checklist and Key with Natural History Notes. Edition Chimaira, Frank- furt am Main. 260 pp. DEUVE, J. 1970. Serpents du Laos. Memoire No. 39. O.R.S.T.O.M., Paris, France. 251 pp. FRANKE, J., AND T. M. TELECKY. 2001. Reptiles as Pets: An Examination of the Trade in Live Reptiles in the United States. The Humane Society of the United States, Washington, D.C. 146 pp.

Herpetological Review 37(2), 2006 173 more than one month were considered not to be preferred species, Herpetological Review. 2006, 37(2), 174-176. (0 2006 by Society for the Study of Amphibians and Reptiles and thus were excluded from the reference species to which fecal samples were compared. Identification of Molluscan Prey from Feces of Prior to its use in the preference experiment, and before it had Iwasaki's Slug Snake, Pareas iwasakii fed in captivity, the same male P. iwasakii excreted feces on 21, 22, and 24 May. These three fecal samples were preserved in 99% ethanol Microscopic examination of the earliest sample revealed MASAKI HOSO* Department of Southeast Asian Area Studies, Kyoto University a jaw and several fragments of a radula that were seemingly de- Kyoto 606-8501, Japan rived from a single snail. The radula and jaw are the hardest parts of a snail's body, except for the shell, and are not digestible be- and cause they are composed of chitin. The minute radular teeth and MICHIO HORI Department of Biological Science, Kyoto University jaw have been used as characters for the classification of terres- Kyoto 606-8502, Japan trial snails (Solem 1978). We examined the radula (using SEM) and the jaw (using a binocular microscope), and compared them Present address/corresponding author: with reference specimens prepared from terrestrial snails from Department of Biological Science, Kyoto University, Kyoto 606-8502, Japan Iriomote Island that had been selected as described above. e-mail: [email protected] The literature survey revealed that the large terrestrial molluscs of Iriomote and Ishigaki include 13 species of snails in 9 genera For decades, herpetologists have been striving to reveal the for- and no native slugs (Table 1). Of the snails, Pareas iwasakii ate aging ecology of snakes (Arnold 1993). In spite of considerable only 7 species (3 genera; Satsuma, Aegista, and Acusta). effort to collect snake dietary information, the diets of many spe- Bekkochlamys masakii and Videna carthcartae were not eaten by cies remain unknown. The diets of snail-eating snakes are among P iwasakii, but the early deaths of these snails precluded confir- the least well known. Adaptations for extracting snails from their mation of their status as prey species. shells have arisen independently in at least three subfamilies of SEM could not reveal the formula of the radula contained in the colubrid snakes: Pareatinae, Natricinae, and Dipsadinae (Gotz feces because the marginal teeth were damaged; thus, the number 2002; Rossman and Myer 1990; Sazima 1989). When feeding on of transverse radular teeth could not be counted. Nonetheless, the a large snail, the snake inserts its mandibles into the snail's aper- ture, and moves each mandible forward and back to extract the body of the snail. Although snails have been confirmed as the pri- mary prey of these snake species via feeding observations of cap- tives and from gut content analysis of wild snakes, the natural prey have rarely been identified to species. This is because mol- luscs that are swallowed without shells were thought to leave no diagnostic remnants in the guts or feces of snakes (Peters 1960). The only available report in which snails eaten by wild snakes were identified to species is that of Judd (1954). He identified prey on the basis of shells, jaws, and radulae of snails found in the stomachs, intestines, and feces of several individuals of Storeria dekayi in Ontario, but he did not describe the characters on which he based his identification of species. All species of the pareatine genus Pareas occur in Southeast Asia and are believed to feed exclusively on terrestrial snails and slugs (Greene 1997). This diet was confirmed by providing non- native terrestrial snails to captive snakes of the genus Pareas (Hirata and Ota 1993; Otani 1983) but the species identity of native prey species has not been determined. Here we present the first record of prey species identified from the feces of Pareas iwasakii, de- termined by scanning electron microscopy (SEM). We propose that our method may be applicable to the identification of prey in other snail-eating snakes. We surveyed literature on the terrestrial molluscan fauna of Iriomote and Ishigaki Islands (Azuma 1995; Habe and Chinen 1974) to determine which species of large snails (shell diameters > 10 mm) and slugs were potential prey for wild P iwasakii,. To FIG. 1. The SEM photographs of the radulae of terrestrial snails. (A) examine preferences for these species of snails, a male P. iwasakii The radula recovered from feces of a wild-captured Pareas iwasakii. (B) (SVL = 440 mm, captured on Iriomote Island on 19 May 2004) The radula of Satsuma caliginosa. (C) The radula of S. yaeyamensis. (D) was maintained in a cage with snails collected from Iriomote Is- The radula of Acusta tourannensis. (E). The radula of Aegista mackensii. land. Snails that remained alive in the cage with the snake for Scale bars = 10 gm.

174 Herpetological Review 37(2), 2006 TABLE 1. The large-shelled native inland snails of Iriomote and Ishigaki Islands (diameter > 10 mm), and feeding preference of Pareas iwasakii for these snails. D: diameter of snails according to Azuma (1995), Y: eaten by the snake, N: not eaten by the snake.

Species D (mm) Feeding result

Acusta tourannensis 16.0 Y Aegista mackensii 42.5 Y Ae. vermis 28.5 Y Ae. caerulea 23.0 Y Ae. osbeckii 20.0 Y Bekkochlamys masakii 15.0 N? Cyclophorus turgidus radians 26.5 N Cyclotus taivanus peraffinis 15.0 N Leptopoma nitidum 17.0 N Luchuena eucharista 11.5 N Satsuma caliginosa 37.0 Y S. yaeyamensis 31.0 Y Videna carthcartae 16.5 N?

diagnostic feature of a reproductive organ would rarely be recov- ered from the stomach because any soft tissues of a snail would be rapidly digested. Moreover, not all stomach contents may be col- lected by forced regurgitation. P. iwasakii is a vulnerable species (Ota 2000), and Pareatinae occur only in the tropical forests of southeastern Asia, which are rapidly being modified by human activities (Laportaferreira and Salomao 2004). Thus, the identifi- cation of prey species of snail-eating snakes based upon fecal analy- sis is strongly recommended as a non-invasive method. To identify prey from visually unrecognizable remains in preda- tor diets, a polymerase chain reaction (PCR)-based molecular

FIG. 2. Binocular photographs of the jaws of terrestrial snails. (A) The method may provide a powerful tool (Symondson 2002). How- jaw recovered from feces of Pareas iwasakii. (B) The jaw of Satsuma ever, molecular methods have three disadvantages in comparison caliginosa. (C) The jaw of S. yaeyamensis. Scale bars = 1 mm. with our method. First, molecular methods require more expen- sive equipment and reagents than does our method. Second, PCR is not always successful because of the possible deterioration of radula was conclusively identified as belonging to an individual DNA during digestion. Third, molecular methods may not reveal of the genus Satsuma based on the diagnostic deeply notched tri- the number and size of identified prey. In the present paper, the dent structure of the radular tooth (Figs. 1A—E). Although it was number of prey consumed was confirmed to be one individual. difficult to distinguish between the radular teeth of S. caliginosa Although the size of the prey was not definitively determined, and S. yaeyamensis (Figs. 1B, C), the two congeners could be dis- examination of reference specimens enabled us to estimate the tinguished by the size and microstructure of the jaw (Figs. 2B, C). size of the consumed snail. On the other hand, a disadvantage of Consequently, the prey item was identified as S. caliginosa (Figs. our method is that it requires preparation of reference samples of IA, B, 2A, B). Furthermore, comparison of the recovered jaw with radulae of local snails. However, molecular methods also require those of S. caliginosa of various sizes suggests that the prey item the preparation of reference DNA sequences, although sequences was an immature individual. from closely related species in DNA databases would also yield Food habits of snakes are generally investigated by examina- useful results. We suggest that the identification of radulae and tion of gut contents of preserved specimens or by forced regurgi- jaws from feces is a more useful method for identifying the prey tation of stomach contents from live specimens. Because both of snail- and slug-eating snakes than is identification from DNA forced regurgitation of soft bodied prey and identification of ex- contained in feces or identification from stomach contents obtained tracted or well-digested snails are difficult, most herpetologists by sacrificing snakes. Furthermore, the stomach contents of road- have thought that it is impractical or impossible to identify mol- killed snakes or preserved snake specimens in museums may be luscan prey eaten by snakes (Cobb 2004; Kofron 1982; Peters available. 1960). Conceivably, the stomach contents of a snake that had eaten We offer the following suggestions to those preparing samples a snail just before capture might include the undigested reproduc- for the identification of molluscan prey based on radulae retained tive organ, which is an important diagnostic feature. However, the in stomach or fecal contents of snakes. It is best to clean the radu-

Herpetological Review 37(2), 2006 175 lae in vials with very little water, using ultrasonic cleaning equip- KOFRON, C. P. 1982. A review of the Mexican snail-eating snakes, Dipsas ment for a few minutes. Excessive treatment sometimes disinte- brevifacies and Dipsas gaigeae. J. Herpetol. 16:270-286. grated the fragile radulae of snails collected from feces. Holznagel LAPORTAFERREIRA, I. L., and M. D. G. SALOMAO. 2004. Reptile predators (1998) recommended an enzymatic cleaning method for radulae of terrestrial gastropods. In G. M. Barker (ed.), Natural Enemies of that could be adapted for use with fecal samples. Because it is Terrestrial Molluscs. Vol. 8, pp. 427-482. CABI Publishing, Wallingford. difficult to distinguish the dorsal from the ventral side of a radula OTA, H. 2000. Iwasaki's slug snake. In Environment Agency of Japan prior to SEM, the radulae should be divided into two or more pieces (ed.), Threatened Wildlife of Japan -Red Data Book 2nd ed.- REP- and the resulting samples prepared so that both sides will be vis- TILIA I AMPHIBIA, p. 58. Japan Wildlife Research Center, Tokyo. ible during SEM observation. OTANI, T. 1983. An additional record of the snake Pareas iwasakii from Our captive observations suggest that P iwasakii has prefer- Iriomote Jima, the Yaeyama Islands with some notes on its feeding ences for specific groups of land snails. Otani (1983) reported that habits. Akamata 1:8-11. P. iwasakii refused to eat certain snails including Cyclophoracea PETERS, J. A. 1960. The snakes of the subfamily Dipsadinae. Univ. Michi- (Leptopoma nitidum, Cyclophorus turgidus radians, and Cyclotus gan Mus. Zool., Misc. Publ. No. 144. ROSSMAN, D. A., and P. A. MYER. 1990. Behavioral and morphological taivanus peraffinis). Because of the early accidental death of adaptations for snail extraction in the North American brown snakes Bekkochlamys masakii during our experiment, it is unclear whether (genus Storeria). J. Herpetol. 24:434-438. P iwasakii will consume that species. SAZIMA, I. 1989. Feeding behavior of the snail eating snake, Dipsas in- The identified prey, S. caliginosa, is one of the most abundant, dica. J. Herpetol. 23:464-468. rather arboreal snails in the habitat of P. iwasakii at night (M. Hoso, SOLEM, A. 1978. Classification of the land mollusca. In V. Fretter and J. unpubl.). The snake is assumed to be an arboreal, forest-depen- Peake (eds.), Systematics, Evolution and Ecology. Vol. 2A, pp. 49-97. dent species (Toyama 1996), and several species of Pareas are Academic Press Inc., London. able to extract snails deftly and eat soft tissues while perched on a SYMONDSON, W. 0. C. 2002. Molecular identification of prey in predator branch in captivity (Gotz 2002; M. Hoso, pers. obs.). These obser- diets. Mol. Ecol. 11:627-641. TOYAMA, M. 1996. Typhlopidae and Colubridae. vations suggest that P. iwasakii forages above the ground, so we In S. Sengoku, T. Hikida, M. Matsui, and K. Nakaya (eds.), Amphibians, Reptiles, suppose that the snake encounters S. caliginosa frequently. Chondrichthyes. Vol. 5, pp. 83-96. Heibonsya, Tokyo.

Acknowledgments.-We thank S. Kobayshi, M. Saigusa, and H. Yam for providing facilities in the field, and C. Matsumoto and H. Ota for providing useful information. We also thank Y. Takami and E. Takahashi for teaching us the use of SEM. We thank A. Mori and A. Savitzky for Artwork and Slides Wanted for HR valuable comments on the manuscript. We are indebted to A. Iwata for supporting our research. This work was partly supported by grants-in-aid from the Science Research on Priority Areas (1408703) and the Grant for We are always interested in obtaining illustra- the Biodiversity Research of the 21st Century COE (A14) of the Japan tions of herpetological subjects for publication in Ministry of Education, Culture, Sports, Science and Technology to M. Herpetological Review Generally, original drawings Hori. This paper is a contribution from Iriomote Station, University of should be of a scale that would permit reduction to the Ryukyus. fit a 90-mm wide column. Original art, or high qual- LITERATURE CITED ity photocopies, should be packaged to ensure safe delivery and sent to the Editor. Alternatively, we ARNOLD, S. J. 1993. Foraging theory and prey-size-predator-size relations would be pleased to receive material in electronic in snakes. In R. A. Seigel and J. T. Collins (eds.), Snakes: Ecology and format; consult the Editor for appropriate file for- Behavior, pp. 87-115. McGraw-Hill, Inc., New York. mats and sizes. AZUMA, M. 1995. Colored Illustrations of the Land Snails of Japan. Also, we are interested in evaluating outstand- Hoikusha Publishing Co., Osaka. COBB, V. A. 2004. Diet and prey size of the flathead snake, Tantilla graci- ing color slides or high-resolution digital images of lis. Copeia 2004:397-402. amphibians and reptiles for possible use on future GOTZ, M. 2002. The feeding behavior of the snail-eating snake Pareas HR covers. When reviewing material for submis- carinatus Wagler 1830 (Squamata: Colubridae). Amphibia-Reptilia. sion, photographers should keep in mind the verti- 23:487-493. cal format of our covers. In addition to highlighting GREENE, H. W. 1997. Snakes: The Evolution of Mystery in Nature. Univ. California Press, Berkeley. outstanding photography, our cover subjects should HABE, T., and M. CHINEN. 1974. Land molluscan fauna of Ishigaki and lend themselves to communication of biologically Iriomote Islands, with notes on biogeography of Ryukyu Archipelago. interesting information through accompanying text. Mem. Natio. Sci. Mus. 7:121-128, pl. 15-17. If you would like to have your work considered, HIRATA, T., and H. OTA. 1993. Predation on snails by the pareatine snake please contact the Editor prior to sending any ma- Pares iwasakii. Jap. J. Herpetol. 15:90-91. HOLZNAGEL, W. E. 1998. Research note: A nondestructive method for clean- terial. We prefer to review images as JPEG or PDF ing gastropod radulae from frozen, alcohol-fixed, or dried material. files before requesting examination of original slides. Am. Malacol. Bull. 14:181-183. Postal and e-mail addresses are listed on the in- JUDD, W. W. 1954. Observations on the food of the little brownsnakes, side front cover. Storeria dekayi, at London, Ontario. Copeia 1954:62-64.

176 Herpetological Review 37(2), 2006 hibit long-term social and genetic monogamy, with E. cunninghami TECHNIQUES and E. stokesii exhibiting strong inbreeding avoidance (Gardner et al. 2001, 2002; O'Connor and Shine 2003; Stow et al. 2001; Stow and Sunnucks 2004a,b). Such behavioral traits are extremely Herpetological Review, 2006, 37(2), 177- 180. © 2006 by Society for the Study of Amphibians and Reptiles rare in lizards (Bull 2000), therefore the genus provides a unique opportunity to examine the evolution of complex sociality within Cross-Species Amplification of DNA Microsatellite squamate reptiles and test existing hypotheses about the evolution Loci in an Australian Lineage of Social Lizards of vertebrate sociality (Chapple 2003). (Scincidae, Genus Egernia) Several factors have enabled Egernia to be utilized as a 'model' system for examining the evolution of sociality and monogamy in lizards. First, most species are large, long-lived and exhibit a strong DAVID G. CHAPPLE* attachment to a home site (e.g., rock crevice, burrow, log) and School of Botany and Zoology, Australian National University Canberra, Australian Capital Territory 0200, Australia therefore are well-suited to long-term behavioral and genetic stud- ies (Chapple 2003). Second, a large number of microsatellite prim- Allan Wilson Centre for Molecular Ecology and Evolution School of Biological Sciences, Victoria University of Wellington ers have been developed for E. stokesii (Gardner et al. 1999; 11 PO Box 600, Wellington, New Zealand EST primers), E. cunninghami (Stow 2002; 5 Ecu primers) and e-mail: [email protected] the closely related Tiliqua rugosa (Cooper et al. 1997; 6 Tr prim- ers). However, what has facilitated the research to date is a high ADAM J. STOW Department of Biological Sciences, Macquarie University degree of successful cross-species PCR amplification of New South Wales 2109, Australia microsatellite loci within the lineage using the same primer pairs. e-mail: [email protected] These primers have now been utilized for examining aspects of sociality and mating systems in E. stokesii (Gardner et al. 2001, DAVE O'CONNOR E. saxatilis School of Biological Sciences, University of Sydney 2002), (O'Connor and Shine 2003), E. cunninghami New South Wales 2006, Australia (Stow et al. 2001; Stow and Sunnucks 2004a,b), E. striolata e-mail: [email protected] (Bonnett 1999; Bull et al. 2001), E. whitii (Chapple and Keogh 2006) and E. frerei (Fuller et al. 2005). SUSAN FULLER School of Biological Sciences, Flinders University Because this is a burgeoning and active area of research, similar GPO Box 2100, Adelaide, South Australia 5001, Australia studies are expected to be conducted for most other Egernia spe- School of Natural Resource Sciences, Queensland University of Technology cies (Chapple 2003). However, at present there is a lack of de- GPO Box 2434, Brisbane, Queensland 4001, Australia tailed information on the cross-species amplification of available e-mail: [email protected] Egernia microsatellite primers (especially for those that were prob- and lematic for some species). Microsatellite markers are costly and MICHAEL G. GARDNER time intensive to develop and successful cross-species amplifica- School of Biological Sciences, Flinders University tion can represent a substantial reduction in cost and time. De- GPO Box 2100, Adelaide, South Australia 5001, Australia tailed information on the potential utility of each microsatellite e-mail: [email protected] locus would aid in selecting primers for particular Egernia spe- *Corresponding author cies, saving time and money. Here we report the cross-species application of the available microsatellite primers in five Egernia Advances in molecular technology have led to the discovery of species. These species represent four of the six species groupings a number of lizard species living in stable family groups. These within the genus: E. cunninghami and E. stokesii (cunninghami social lizards provide an opportunity to test current hypotheses group, 4 species), E. saxatilis (striolata group, 9 species), E. whitii about the evolution and maintenance of vertebrate social systems (whitii group, 12 species) and E. frerei (major group, 4 species). developed using endothermic models (i.e., birds and mammals). Since the remaining two species groups comprise a total of three As such, social lizards have increasingly been the focus of intense species (E. kingii group, 1 species; E. luctuosa group, 2 species), research, in particular studies within the Australian Egernia Group the four species groups that we examine contain 29 of the 32 spe- lineage of skinks. cies in the genus and therefore should provide valuable informa- The scincid genus Egernia comprises 32 species of medium to tion for future researchers. In order to supplement our detailed large-sized viviparous skinks and is endemic to Australia (Chapple analysis of five species we provide a brief summary of the results 2003). Recently, much interest has been generated in this group from published studies that have used microsatellites in Egernia due to the realization that complex social systems and monogamy species. are widespread within the genus, with reports on 26 of the 32 spe- The PCR conditions used for each species are contained within cies suggesting social structures of varying complexity, ranging the original published studies and therefore we only provide rel- from predominately solitary through to large social aggregations evant information that has not been published elsewhere. For E. in excess of 30 individuals (reviewed in Chapple 2003). Long- stokesii details regarding PCR conditions and parameters are con- term behavioral and genetic studies on four species have shown tained in Cooper et al. (1997) and Gardner et al. (1999, 2000, 2001, that these large aggregations are stable between years and consist 2002), with the conditions for the Ecu primers as detailed in Stow of closely related individuals (Chapple 2003). In addition, E. (2002). For E. cunninghami this information is provided in Stow cunninghami, E. saxatilis and E. stokesii have been shown to ex- et al. (2001), Stow (2002), and Stow and Sunnucks (2004a, b),

Herpetological Review 37(2), 2006 177 TABLE 1. Cross-amplification of the available microsatellite primers in the Egernia, Tiliqua and Cyclodomorphus species tested to date. P = poly- morphic; M = monomorphic; U = unsuccessful amplification; ? = some degree of amplification (see Table 2 for further detail). Species codes as follows: EC = E. cunninghami, EF = E. frerei, El = E. inornata, ESa = E. saxatilis, ESto = E. stokesii, EStr = E. striolata, EW = E. whitii, TA = T adelaidensis, TR = T rugosa, TS = T scincoides, CB = C. branchialis, CC = C. casuarinae, CG = C. gerrardii. Locus EC EF EI ESa ESto EStr EW TA TR TS CB CC CG Tr3.2 P U' P P4 U P' P6 P' P' U' Tr4.6 M?' P' M?' P6 M?' P' U' Tr4.11 P M P pl U P' PS M?' P' U' Tr5.20 P P P P' U U' P' P' M?' Tr5.21 P P P pl U P' P" P' P' U' EST1 P P P P2 P EST2 P P' P2 P5 P P' P' P' EST3 U U' P2 U' U U' U' U' EST4 P P?' P2 U' U 1 M?' EST6 P' EST8 9 P' P' Ps U M?' M?' M?' EST9 P P P' ? 2 U' U P' U' P' EST12 P M ?2 Ps P EST14 7 EST15 U EST16 Ecul P P3 P Ecu2 P 133 P Ecu3 P U U3 U Ecu4 P U P3 U P? EcuS P P P3 P P?

References 'Gardner (1999); 2Gardner et al. (1999, 2000); 3Stow (2002); 4Bull et al. (2001); 5Bonnett (1999); 6Cooper et al. (1997). while those for E. frerei are contained in Cooper et al. (1997), are contained in Cooper et al. (1997), Gardner et al. (1999), and Gardner et al. (1999, 2000), Stow (2002), and Fuller et al. (2005). O'Connor and Shine (2003). PCR was performed in a 10 gl vol- Information relating to the cross-amplification in E. whitii is ume reaction, containing 5 gl of template DNA, lx PCR reaction contained in Chapple and Keogh (2005, in press). In this species buffer, 1.5 mM MgC12, 0.1 mM dNTPS, 400 nM of each primer, PCR was performed in a 20 gl volume reaction, containing ap- and 0.25 units of Taq DNA polymerase (all Sigma reagents). The proximately 100 ng of template DNA, 2.5 pmol of the M13(-21) PCR parameters for the Ecu primers followed Stow (2002). The tailed sequence-specific forward primer, 10 pmol of the sequence- PCR program for EST2, EST12 and Tr4.11 involved an initial specific reverse primer, 10 pmol of a fluorescent dye-labelled denaturing step of 94°C for 3 min, followed by 35 cycles of 94°C M13(-21) universal primer (either 6-FAM, NED or PET; Applied for 30 sec, 55°C for 30 sec, and 72 °C for 30 sec, followed by a Biosystems), 2 gl 10x PCR Buffer, 2 ill 10x Enhancer Solution final extension step at 72°C for 10 min. The parameters for EST1 (Gibco BRI Life Technologies), 3 mM MgSO 4, 2 mM dNTPs and and Tr5.21 were similar except that the 35 cycles were substituted 0.2 units of Platinum Taq DNA polymerase (Gibco BRI Life Tech- with one cycle with an annealing temperature of 55°C, followed nologies). A 'stepping down' PCR program was used to amplify by single cycles at 53 °C, 51°C, and 49°C, with a further 30 cycles each locus. Reactions were initially denatured at 94°C for 5 min, at 47°C. For Tr5.20, these cycles were replaced by 7 cycles at followed by an annealing step at 70 °C for 15 sec and extension at each of the following annealing temperatures: 55°C, 53°C, 51 °C, 72°C for 1.5 min. This was followed by a further round of dena- and 49°C. For EST8 and EST9 the 35 cycles were replaced by turation at 94°C for 30 sec, annealing at 70°C for 15 sec and ex- annealing temperatures of 55 °C (2 cycles), 53°C (2 cycles), 51 °C tension at 72°C for 1.5 min. The annealing temperature was then (2 cycles), 49°C (2 cycles) and 47°C (20 cycles). dropped by 5°C in the next two rounds of cycling. This 'stepping Within Egernia there are eight primer pairs that appear to per- down' in annealing temperature was repeated until a final anneal- form well across all lineages (Table 1). The most successfully used ing temperature of 35°C was reached. The next 50 cycles then loci are EST1, EST2, EST12, Tr5.20 and Tr5.21 with promising were performed with this annealing temperature. A final exten- preliminary results from the recently developed Ecul, Ecu2 and sion step at 72°C was done for 7 min. EcuS primers. The Egernia range of primers is highly polymor- Details concerning the amplification of the loci in E. saxatilis phic and extremely informative (Table 2). Tr4.11 however, ap-

178 Herpetological Review 37(2), 2006 TABLE 2. Cross-amplification of the available microsatellite primers in five Egernia species. Several loci were not trialled in each of the species: E. cunninghami (Tr3.8, EST14, EST16), E. saxatilis (Tr3.2, Tr3.8, Tr4.6, EST3-4, EST6, EST14-16), E. whitii (Tr3.8, Tr4.6, EST6, EST14-16), E. frerei (Tr3.2, Tr3.8, EST3, EST6, EST14-16, Ecu2, Ecu4-5) and E. stokesii (Ecu3). N = number of individuals trialled; NA = number of alleles; Ho = Observed heterozygosity; He = Expected heterozygosity; HWE P = P-value for Hardy-Weinberg equilibrium for each locus.

Locus N Size range NA Ho He HWE P Notes

'E. cunninghami Tr3.2 189 161-269 20 0.961 0.928 NS Linkage disequilibrium with EST12 Tr4.6 10 amplified NS Could not optimise '(47, 55, 60) Tr4.11 189 amplified 12 0.460 0.809 < 0.001 Sex-linked (females heterozygous) Tr5.20 189 146-152 3 0.260 0.268 NS High frequency of putative null alleles at one site (ca. 12%) Tr5.21 189 79-145 18 0.887 0.875 NS EST1 189 209-337 20 0.940 0.883 NS EST13 (= EST2) 189 164-252 17 0.868 0.926 NS EST4 189 amplified 17 0.538 0.888 < 0.001 High frequency of null alleles (ca. 24%) EST6 10 amplified NS Could not optimize '1(47, 55, 60) EST8 10 amplified NS Could not optimize '1(47, 55, 60) EST9 189 215-279 12 0.660 0.646 NS EST12 189 amplified 26 0.915 0.921 NS Linkage disequilibrium with Tr3.2 Ecul 161 144-194 17 0.883 0.902 NS Ecu2 161 154-196 13 0.848 0.843 NS Ecu3 161 220-272 11 0.784 0.825 NS Ecu4 161 76-288 29 0.825 0.924 NS null alleles (ca. 9%) EcuS 161 120-164 17 0.894 0.904 NS E. saxatilis Tr4.11 28 134-137 2 Sex-linked Tr5.20 277 127-191 29 0.693 0.894 <0.001 null alleles (ca. 13%) Tr5.21 280 80-120 17 0.821 0.883 NS EST1 279 209-309 25 0.928 0.931 NS EST2 276 194-274 21 0.804 0.922 NS EST8 42 Amplified Could not optimize EST9 24 Amplified Could not optimize EST12 281 191-267 19 0.865 0.606 NS Ecul 4 Amplified Ecu2 4 Amplified EcuS 4 Amplified E. whitii ESTI 127 226-314 20 0.787 0.928 NS EST2 127 188-280 20 0.969 0.923 NS EST4 127 123-179 14 0.858 0.874 NS EST9 15 259-277 4 0.400 0.579 NS Putative null alleles EST12 127 276-374 21 0.890 0.933 NS Ecul 16 159-243 18? 0.813 0.915 NS Di-repeat with 4 peak stutter Ecu2 127 149-179 13 0.535 0.790 <0.001 null alleles (ca. 24%) EcuS 14 122-136 4 0.929 0.643 NS E. frerei Tr4.6 1 Amplified 1 Tr4.11 28 130 I N/A N/A N/A Monomorphic Tr5.20 229 118-126 6 0.489 0.485 NS Di-repeat with stutter Tr5.21 224 82-88 7 0.799 0.754 NS Alleles differ by lbp ESTI 225 188-266 30 0.804 0.935 NS Alleles differ by 2bp EST2 229 173-243 18 0.764 0.912 <0.001 Short allele dominance EST4 225 108-120 4 0.280 0.261 NS EST8 85 104-176 ? Could not optimize EST9 228 227-303 20 0.803 0.904 NS EST12 29 247 1 Monomorphic Ecul 16 141-151 3? Could not optimize Ecu3 15 229-281 9 0.800 0.883 NS E. stokesii Tr3.2 150 176-234 20 0.867 0.880 NS Linkage disequilibrium with EST12 Tr5.21 50 4 0.292 0.2 0.0012 Null alleles (ca 13%), stutters ESTI 150 234-282 12 0.913 0.865 NS EST2 141 206-286 24 0.915 0.910 NS

Herpetological Review 37(2), 2006 179 TABLE 2. Continued.

Locus N Size range NA Ho He HWE P Notes

EST3 141 246-346 16 0.865 0.884 NS Low frequency of null alleles, short allele dominance EST4 150 141-189 11 0.793 0.850 NS Low frequency of null alleles, short allele dominance EST6 10 163-189 8 0.80 EST8 149 101-141 8 0.799 0.816 NS EST9 10 235-263 9 0.60 High frequency of null alleles EST12 149 288-336 13 0.846 0.879 NS Linkage disequilibrium with Tr3.2 EST14 10 114-178 7 0 May contain null alleles EST15 10 129-141 2 0.10 EST16 10 156-184 5 0.40 May contain null alleles Ecul 50 111-186 4 0.612 0.22 <0.001 Null alleles (ca 28%), large alleles stutter. Ecu2 50 132-170 8 0.683 0.62 NS Ecu4 50 64-72 3 0.578 0.58 NS EcuS 50 107-121 11 0.87 0.8 NS

1 PCR conditions for optimized loci given in Stow et al. (2001) and Stow (2002) '' = annealing temperatures trialled (V) pears to be sex linked (females heterozygous) and Tr3.2 and EST12 COOPER, S. J. B., C. M. BULL, AND M. G. GARDNER. 1997. Characterization appear to be directly linked (Table 2). Problems with short allele of microsatellite loci from the socially monogamous lizard Tiliqua rug- dominance and null alleles are present within the lineage but are osa using a PCR based isolation technique. Mol. Ecol. 6:793-795. not consistently related to a particular primer but rather the primer- FULLER, S. J., C. M. BULL, K. MURRAY, AND R. J. SPENCER. 2005. Cluster- ing of related individuals in a population of the Australian lizard, Egernia species interaction (Table 2). Overall, the Egernia microsatellite frerei. Mol. Ecol. 14:1207-1213. loci are highly polymorphic and extremely informative for stud- GARDNER, M. G. 1999. A genetic investigation of sociality in the Austra- ies of sociality. lian group liviing lizard Egernia stokesii. Unpublished PhD thesis, Flinders University, Adelaide, Australia. Acknowledgments.-DGC thanks Chris Hayes for assistance in the labo- , C. M. BULL, AND S. J. B. COOPER. 2002. High levels of genetic ratory. Funding was provided to DGC by Australian Geographic, Austra- monogamy in the group-living Australian lizard, Egernia stokesii. Mol. lian Society of Herpetologists, ASIH Gaige Fund, Peter Rankin Trust Fund Ecol. 11:1787-1794. for Herpetology, Joyce W. Vickery Scientific Research Fund, Ecological , AND G. A. DUFFIELD. 2000. Microsatellite muta- Society of Australia and SSB Award for Graduate Student Research. Fund- tions in litters of the Australian lizard Egernia stokesii. J. Evol. Biol. ing for the E. frerei research was provided by the Australian Research 13:541-550. Council and a Program Grant from the Faculty of Science and Engineer- , AND . 2001. Genetic evidence for a fam- ing, Flinders University. SF thanks staff from the Evolutionary Biology ily structure in stable social aggregations of the Australian lizard Egernia Unit of the South Australian Museum for helpful advice and discussion. stokesii. Mol. Ecol. 10:175-183. DO'C thanks Kellie Palmer for assistance in the testing and optimization , S. J. B. COOPER, C. M. BULL, AND W. N. GRANT. 1999. Isolation of of the E. saxatilis primers. Funding was provided to DO'C by a Royal microsatellite loci from a social lizard, Egernia stokesii, using a modi- Zoological Society of New South Wales Ethel Mary Read grant and a fied enrichment procedure. J. Hered. 90:301-304. Peter Rankin Trust Fund for Herpetology grant. O'CONNOR, D., AND R. SHINE. 2003. Lizards in 'nuclear families': a novel reptilian social system in Egernia saxatilis (Scincidae). Mol. Ecol. LITERATURE CITED 12:743-752. STOW, A. J. 2002. Microsatellite loci from the Cunningham's Skink BONNETT, M. P. 1999. The ecology, behaviour and genetic relationships of (Egernia cunninghami). Mol. Ecol. Notes 2:256-257. a population of Egernia striolata. Unpublished Honours Thesis, Hinders , AND P. SUNNUCKS. 2004a. High mate and site fidelity in Cunning- University, Adelaide, Australia. ham's skinks (Egernia cunninghami) in natural and fragmented habi- BULL, C. M. 2000. Monogamy in lizards. Behay. Processes 51:7-20. tat. Mol. Ecol. 13:419-430. , C. L. Griffin, M. Bonnett, M. G. Gardner, and S. J. B. Cooper. , AND . 2004b. Inbreeding avoidance in Cunningham's 2001. Discrimination between related and unrelated individuals in the skinks (Egernia cunninghami) in natural and fragmented habitat. Mol. Australian lizard Egernia striolata. Behay. Ecol. Sociobiol. 50:173- Ecol. 13:443-447. 179. , D. A. BRISCOE, AND M. G. GARDNER. 2001. The impact of CHAPPLE, D.G. 2003. Ecology, life-history, and behavior in the Australian habitat fragmentation on dispersal of Cunningham's skink (Egernia scincid genus Egernia, with comments on the evolution of complex cunninghami): evidence from allelic and genotypic analyses of sociality in lizards. Herpetol. Monogr. 17:145-180. microsatellites. Mol. Ecol. 10:867-878. , AND J. S. KEOGH. 2005. Complex mating system and dispersal patterns in a social lizard, Egernia whitii. Mol. Ecol. 14:1215-1227. , AND . Group structure and stability in social aggregations of White's skink, Egernia whitii. Ethology 112:247-257.

180 Herpetological Review 37(2), 2006 each trap was labeled by array number and type. For example, Herpetological Review, 2006,37(2), 181-183. 2006 by Society for the Study of Amphibians and Reptiles type "A" (PVC), type "B" (coverboard), and type "C" (PC) in array 15 were labeled as "15A," "15B," and "15C," respectively. Innovative Techniques for Sampling Arrays were checked, in order, from the downstream end (array Stream-inhabiting Salamanders 20) to the upstream end (array 1). The PVC trap consisted of a 73 cm section of 18 cm polyvinyl chlorate (PVC) with holes drilled on each end for "stakes" (wire THOMAS M. LUHRING and insulation supports) to anchor it into the spring bed. These were CAMERON A. YOUNG placed in the middle of the spring and were checked internally Savannah River Ecology Laboratory, Drawer E before being rolled over on each survey date. Coverboards were Aiken, South Carolina 29803, USA composed of a 73 cm x 73 cm section of 11-mm plywood. These e-mail: [email protected] e-mail: [email protected] were placed parallel to the spring with approximately 18 cm of the board overhanging the spring or in the spring to mimic the amount of overhang associated with the PC trap. Although salamanders are excellent indicators of environmen- The novel trap, PC, was a "PVC-coverboard hybrid" made of tal health, the ability to catch them efficiently without substan- two independent parts (Fig. 1). The first part of the PC trap was a tially disrupting their habitat is not always practical or even pos- section of 11 mm plywood identical to that of the coverboards. sible with current techniques. Ripping open logs and raking leaf This was used in conjunction with a 73 cm section of lengthwise- packs onto shore (Bruce 1972) are examples of such practices that halved 18 cm PVC. Wire screen (gutter guard) was cut to fit each are disruptive but widely used by herpetologists who have no other end of the PVC halves and was attached to the pipe with six zip means of efficient collection. Drift fences with pitfall traps are ties that were drawn through six small, evenly spaced holes on effective in catching animals moving within or between habitats each end of the PVC. The halved PVC of PC traps were anchored but are time consuming and require an initial financial investment with the same "stakes" as PVC traps. These halved pipes were and constant upkeep to maintain functionality and prevent animal anchored by pushing the "stakes" into the ground on the terres- fatalities (Gibbons and Semlitsch 1981). One current alternative trial side of the pipe and bending their ends to catch the lip of the to drift fences is the use of coverboards (Grant et al. 1992), which pipe. The pipe halves of PC traps were placed inside the spring require less maintenance and sampling effort than drift fences. parallel to and touching the bank with their terrestrial edge as tightly However, coverboards do not integrate captures over a long time fitting with the bank as possible. The terrestrial side was always period and often result in a lower number of captures per trap somewhat lower than the spring side, which allowed salamanders (Grant et al. 1992). to enter and leave at will. While it was possible for the salamanders The purpose of our study was to evaluate the effectiveness of a to navigate in and out of the halved pipe, once they fled to the new trap design for sampling stream-inhabiting salamanders. The water, they would remain motionless in the bottom of the pipe and traps were designed to be non-destructive to the habitat while be- would not move unless disturbed. The plywood was then placed ing economical and time efficient. The new trap, a combination of on the bank with enough overhang (18 cm) to cover the halved- PVC pipe and coverboard (PC trap), was specifically designed to pipe. After lifting the board in the same manner as used for take advantage of the tendency of species in habitats near water to coverboards, the inside of the pipe was examined for salamanders run toward the water when their cover is disturbed and then re- and, if present, the salamanders could be lifted out of the water main motionless amongst the detritus. The coverboard of PC traps while still in the pipe and then could be handled one at time while was used to create a habitat for the salamanders that could be effi- the others remained in the pipe. The space under the pipe was also ciently surveyed and replaced. The other "traps" (coverboards and checked either by sight or, for best results, by using the PVC halve sections of PVC pipe) were used as comparisons. We predicted in place to scoop out anything underneath it. Time and money that the PC trap would have a higher number of captures per trap invested into each trap were estimated by creating a sum of all than coverboards or sections of PVC used separately. Although steps and parts associated with the trap type (cutting boards, con- the techniques tested are being referred to as "traps," they are de- structing half pipes, material prices, etc.). signed to be "escapable" and thus able to be left in place unaltered and unchecked indefinitely without causing mortality to sala- manders or non-target species.

The study site, a seep-fed spring that originated at the bottom of Direction a gorge and terminated at a constructed pond, spanned approxi- coverboard is lifted mately 365 m of mixed hardwood forest near the Fall Line in Rich- mond County, Georgia. Twenty arrays were placed at 12.4-m in- tervals along the spring's banks. An array consisted of one of each trap type placed in random order at 1.8-m intervals. Randomness 1%.".""'""••••■•■.. Coverboard was achieved by designating each trap with a number (PVC = 1, coverboard = 2 and PC = 3) and then using a random number table Halved PVC ■■■ with screen on IS.4.44%.• • Stake to anchor PVC to select their order. Each interval was measured from the closest ends edge of each trap. All traps were 73 cm in length. Arrays were numbered from 1 to 20 starting at the beginning of the spring and FIG. 1. PC trap as used in a spring at the study site.

Herpetological Review 37(2), 2006 181 TABLE 1. Total trap captures of each species and totals for each trap 40 type. Surveys were conducted daily from 24 December 2003 to 3 Janu- ary 3 2004, and on 1 February 2004 and 8 March 2004. 35

Species PVC Coverboard PC traps Total 30 w 25 Larval Pseudotriton ruber 6 16 59 Rl Adult Pseudotriton ruber 0 3 2 2 20 Eurycea guttolineata 0 1 1 3 15 Eurycea cirrigera 0 2 3 5 10 Desmognathus conanti 0 5 0 5 Desmognathus auriculatus 1 1 3 5 5 Eurycea spp. Larvae 0 2 5 7 0 Totals 7 30 73 110 1 3 5 7 9 11 13 Survey Date All traps were set on 22 December 2003 and checked daily from 24 December 2003 to 3 January 2004 and then again on 1 Febru- ary 2004 and 8 March 2004 for a total of 13 days. Traps were —Trap Captures - - Rolling Cover checked, in order, from array 20 to array 1. We recorded the num- ber and developmental stage of each species of salamander seen. FIG. 2. Number of salamanders captured on each survey date (24 De- Prior to release at the site of capture, each salamander was given cember 2003-3 January 2004, 1 February 2004 and 8 March 2004) re- a temporary ID by toe clipping, to keep track of recapture levels sulting from trapping efforts and rolling natural cover present within 1 m and movements. The same data for captures resulting from lifting of the spring. or rolling natural cover within 1 m of the spring were collected, and the location was marked with an orange utility flag bearing information on species, date and time, ID number, and develop- nated under the coverboards and PC traps. Only one snake, a mental stage. Cover that had been lifted or rolled for surveying Nerodiafasciata under trap 17B on 8 March 2004 (ca. 24 cm SVL), purposes was replaced as close to the original position as possible was found during the survey as it occurred during the winter sea- and was checked on each successive survey. All larval salamanders son when they were inactive. Small crayfishes under 15 mm were considered too small for toe clipping were captured, noted, and occasionally found under boards and halved pipes of traps closer released. to the springhead. At the conclusion of trapping, 121 salamanders had been marked The number of trap captures was few at the inception of the and were recaptured 57 times for a total of 178 captures. Species survey in comparison to the number of captures resulting from the captured were Desmognathus auriculatus, D. conanti, Eurycea rolling of natural cover. However, the number of trap captures cirrigera, E. guttolineata, and Pseudotriton ruber. Larval P. ruber increased as the project continued until they accounted for most were captured most frequently (Table 1). Total number of sala- daily captures (Fig. 2). The initial decrease in the numbers of sala- mander captures for the PVC, coverboard, and PC traps were 7, manders in traps and under natural cover may have resulted from 30, and 73, respectively. their refugia being disturbed on a constant basis for the first eleven Each step in construction was done all at once (wood cutting, survey dates. On the last three survey dates, which were each a pipe sawing, etc.) and was timed along with assemblage to esti- month apart, the number of salamanders under each type of refu- mate overall construction times (Table 2). We used a hacksaw to gia increased and the number of salamanders under natural cover cut through the PVC, which added time onto PVC and PC traps. approached pre-survey levels. This positive trend is probably due PVC was the least effective of the three types in terms of both to the decrease in disturbance to refugia as well as a possible in- time and money invested per capture. Coverboards were the most crease in activity. The PC traps were more effective than all other efficient in terms of construction time invested per capture. The traps in terms of total captures and became the most overall pro- most efficient trap in terms of money invested per capture was the ductive method as the study continued. This trend may be the re- PC trap. Recently metamorphed Rana clamitans (N = 9) hiber- sult of salamanders having more time to find the traps and use them as refugia. Forty-six (63%) of the 73 salamanders found in the PC traps were either in or under the PVC, which may indicate TABLE 2. Construction cost and time invested per trap and per capture. a microhabitat preference. A potential bias of the PC trap is that it depends on animals to Construction Construction Cost/ Time/ select it as refugia and may not be equally effective for species Cost Time Capture Capture (min) with differing microhabitat preferences. This bias is evident in the number of the total larval P. ruber that were found in PC traps (N PVC 2.39 4 $6.81 11.4 = 59) as compared to the combined total of coverboards and PVC Coverboard 1.62 5 $1.07 3.3 traps (N = 22). Another possible bias was exhibited by D. conanti, PC Traps 3.22 15 $0.88 4.1 which were present only under coverboards.

182 Herpetological Review 37(2), 2006 One major advantage of the PC trap was that it could be left Herpetological Review. 2006, 37(2). 183-185 unattended for an indefinite amount of time without any mortality (:). 2006 by Society for the Study of Amphibians and Reptiles because the animals were able to escape. This allows much more flexibility in trapping schedule and much less constant upkeep An Effective and Durable Funnel Trap for when compared to techniques such as drift fences that must be Sampling Terrestrial Herpetofauna checked daily (Gibbons and Semlitsch 1981). Although drift fences are an effective way of collecting species moving from one finite JEFFREY R. ROW area to another such as a seasonal wetland, they may not be prac- and tical to use in long and thin habitats such as springs and streams GABRIEL BLOUIN-DEMERS* that cannot be surrounded easily. Drift fences may also fail to Department of Biology, University of Ottawa, 30 Marie-Curie, Ottawa capture salamanders that are able to climb out of pitfalls or over (Ontario), Canada, KIN 6N5 fences. Ryan et al. (2002) suggested that a combination of census * Corresponding author; e-mail: [email protected] techniques should be used when monitoring herpetofaunal com- munities to account for the maximum number of species. The PC The global population decline of amphibians has attracted much trap, while efficient in sampling salamanders in its immediate area international attention (Alford and Richards 1999). Although less and habitat, is not designed to be an all-inclusive, mass sampling attention has been paid to reptile populations, they appear to be technique such as a drift fence. Instead, it is most useful when declining at a faster rate than amphibian populations (Gibbons et sampling fully or semi-aquatic salamanders in or in very close al. 2000). Part of the discrepancy in attention between the two proximity to water on a sporadic sampling schedule. groups is due to the difficulty in estimating the population trends of reptiles. Reptiles tend to be secretive, solitary, and dispersed, Acknowledgments.—We thank J. R. and S. N. Luhring for their finan- making them difficult to sample, and these traits hinder long-term cial support and J. R. Luhring especially for many hours of manual labor. mark-recapture studies that are necessary to generate population We thank the Georgia-Carolina Council of the Boy Scouts of America, P. trends. Patton, and the Powell family for access to the study site. We thank J. W. To study the population ecology of terrestrial herpetofauna, re- Gibbons, J. D. Willson, C. T. Winne, A. E. Liner, and S. H. Schweitzer for searchers have employed various techniques such as timed their review of the manuscript and editing suggestions. We thank S. B. Castleberry, M. R. Boehm, and G. J. Graeter for their comments and in- searches, cover boards, and drift fences with pit fall traps or fun- sight. We thank J. R. Pittard and "the Samanthas" for assistance in the nel traps (Renken et al. 2004; Ryan et al. 2002). Although all of field. The procedures used in this study were approved by the University these methods are effective, drift fences with funnel traps catch of Georgia animal care and use committee (A2003-10024, "Reptile and the largest species diversity and the most individuals (Ryan et al. amphibian research—general field studies"). Manuscript preparation was 2002), especially for reptiles. Here we present our design for an aided by the Environmental Remediation Sciences Division of the Office effective, light, and durable funnel trap. of Biological and Environmental Research, U.S. Department of Energy Species that aggregate for hibernation allow for a unique op- through Financial Assistance Award no. DE-FC09-96SRI8546 to the Uni- portunity to acquire reliable population size estimates (Blouin- versity of Georgia Research Foundation. Demers et al. 2000; Prior et al. 2001). Each spring since 1996, we have sampled a population of Black Ratsnakes (Elaphe LITERATURE CITED alleghaniensis) at the Queen's University Biological Station (150 BRODIE, E. D., JR. 1977. Salamander antipredator postures. Copeia 1977: km S of Ottawa, Ontario, Canada) by enclosing 12-18 hibernacula 523-535. with perimeter fences fitted with funnel traps. We surround the BRUCE, R. C. 1972. The larval life of the red salamander, Pseudotriton hibernaculum with a wooden frame to which we staple heavy- ruber. J. Herpetol. 6:43-51. gauge polyethylene plastic sheeting. We fold the plastic on itself GIBBONS, J. W., AND R. D. SEMLITSCH. 1981. Terrestrial drift fences with twice before stapling and we staple through a piece of cardboard pitfall traps: an effective technique for quantitative sampling of ani- (3 x 10 cm) to prevent the plastic from ripping under wind force. mal populations. Brimleyana 7:1-16. We fold the plastic towards the inside of the fence at the bottom GRANT, B. W., A. D. TUCKER, J. E. LOVICH, A. M. MILLS, P. M. DIXON, AND and pile rocks, sticks, and leaves on the fold until it is completely J. W. GIBBONS. 1992. The use of coverboards in estimating patterns of reptile and amphibian biodiversity. In R. Seigel and N. Scott (eds.). covered. We install one funnel trap along the fence when the en- Wildlife 2001, pp. 379-403. Elsevier Science Publ., Inc. London, closure has a diameter < 5 m and we install two traps diametri- England. cally opposed if the diameter of the enclosure is between 5 and 10 RYAN, T. J., T. PHILIPPI, Y. A. LEIDEN, M. E. DORCAS, T. B. WIGLEY, AND J. m. The enclosure and funnel traps allow a large proportion of the W. GIBBONS. 2002. Monitoring herpetofauna in a managed forest land- ratsnake population to be sampled: we capture ca. 200 individuals scape: effects of habitat types and census techniques. Forest Ecology each spring (Blouin-Demers et al. 2000). We capture all size and Management 167: 83-90. classes, from neonates (SVL = 250 mm, mass = 7 g) to adults (SVL = 1750 mm, mass = 1250 g). In addition, our funnel traps regularly capture (approximately 50 individuals per year) the other eight species of snakes encountered at our study site that use ratsnake hibernacula: Nerodia sipedon, Thamnophis sirtalis, T sauritus, Storeria dekayi, S. occipitomaculata, Diadophis punctuatus, Liochlorophis vernalis, and Lampropeltis triangulum. We construct the cylindrical frame of the trap and the funnel

Herpetological Review 37(2), 2006 183 with 1.27 cm (0.5 inch) hardware cloth (18-20 wire gauge) fas- from pushing their heads through the hardware cloth, we wrap the tened with cage rings (these materials are commonly available at trap and funnel with aluminum window screening. We overlap the farm supply stores). We shape the funnel to be ca. 60 cm long, screening ca. 10 cm and sew it to the hardware cloth with alumi- with a large opening of diameter > 30 cm and a small opening of num wire (20-22 gauge), using 2-cm long stitches. The window diameter ca. 5 cm. We attach the funnel to a square piece (60 cm x screening and hardware cloth can both be purchased in 122 cm (4 60 cm) of 1.91 cm (0.75 inch) plywood with a 30 cm circular hole feet) width, which is a convenient trap length. cut 5 cm from the bottom (Fig. 1). The funnel is thus 5 cm above After sewing the window screening to the trap, we install the the ground after installation, but because we pile rocks, sticks, and leaves on top of the plastic sheeting we use as fencing, the opening of the funnel is actually level with the substrate (Fig. 2). We push the funnel from the outside through the opening in the plywood until it fits tightly and then cut, flatten, and secure (with 8 mm staples) the outer rim of the funnel to the plywood (Fig. 1). The diameter of the trap is also 30 cm, allowing us to use the plywood circle (cut out for the funnel) as the back of the trap. To allow the capture of small snakes and to dissuade larger snakes

T5 cm

1 120 cm

FIG. I. Diagram of a simple and durable funnel trap. The trap (2) and the funnel (3) are made out of 1.27 cm (0.5 inch) hardware cloth and surrounded with aluminum window screening (1). The support for the funnel (5) and the back of the trap (4) are made with 1.91 cm (0.75 inch) FIG. 2. Photographs of the simple and durable funnel trap (A), of the plywood. The funnel is pushed through the hole in the support and then accumulation of debris on the plastic fencing that allows the funnel to be cut, flattened, and secured with staples around the edge of the funnel level with the substrate along a perimeter fence (B), and of the trap in use (arrows). at the end of a metal drift fence with leads (C).

184 Herpetological Review 37(2), 2006 plywood circle at the back of the trap and secure it with 8 mm ENGE, K. M. 1997. Use of silt fencing and funnel traps for drift fencing. staples. It is important to attach the back of the trap last because it Herpetol. Rev. 28:30-31. facilitates sewing the screening and the staples can secure both GIBBONS, J. W., D. E. Scam T. J. RYAN, K. A. BUHLMANN, T. D. TUBERVILLE, the hardware cloth and the mesh to the back of the trap. B. S. METTS, J. L. GREENE, T. MILLS, Y. LEIDEN, S. POPPY, AND C. T. Finally, we coat the small opening of the funnel with two-part WINNE. 2000. The global decline of reptiles, deja vu amphibians. BioScience 50:653-666. epoxy (we found Plasti-Dip to be much less durable than epoxy). PRIOR, K. A., G. BLOUIN-DEMERS, AND P. J. WEATHERHEAD. 2001. Sampling We dip the end of the funnel multiple times to build a thick coat. biases in demographic analyses of black rat snakes (Elaphe obsoleta). This serves two purposes: it makes the funnel more durable by Herpetologica 57:460-469. hardening and securing the window screening and it covers sharp RENKEN, R. B., W. K. GRAM, D. K. FANTZ, S. C. RICHTER, T. J. MILLER, K. ends from the cut window screening which could injure snakes or B. RICKE, B. RUSSELL, AND X. WANG. 2004. Effects of forest manage- deter them from entering the trap. ment on amphibians and reptiles in Missouri Ozark forests. Cons. Biol. The trap is pushed tight against the funnel and secured by at- 18:174-188. taching a rope to the plywood on each side of the funnel (through RYAN, T. J., T. PHILIPPI, Y. A. LEIDEN, M. E. DORCAS, T. B. WIGLEY, AND J. W. GIBBONS. 2002. Monitoring herpetofauna in a managed forest land- two drilled holes) and passing that rope around the back of the scape: effects of habitat types and census techniques. For. Ecol. Man- trap. Because the trap is made of screening and hardware cloth, a age. 167:83-90. visual inspection of the trap is sufficient to detect the presence of animals. When animals are captured, we detach the retaining rope, pull the trap back and quickly surround the opening of the trap with a snake bag (we use a pillow case). We gently raise the back of the trap until the animals slide in the bag. To prevent bites when Herpetological Review. 2006,37(2), 185 - 187. O 2006 by Society for the Study of Amphibians and Reptiles dealing with venomous snakes, one could empty the trap in a hard plastic bucket or a garbage can instead of a bag. Using Deep-Water Crawfish Nets to Capture To prevent overheating, we ensure that animals have shade by Aquatic Turtles covering one end of the trap with a tarp or plywood scraps. Only one end is covered, as covering the whole trap could impede air- flow and also lead to overheating. BRAD M. GLORIOSO and Our funnel trap was inspired by earlier versions that were made MATTHEW L. NIEMILLER solely of window screening held with office staples (e.g., Enge Department of Biology, Middle Tennessee State University 1997), but we found those too flimsy for our purpose. The addi- Murfreesboro, Tennessee 37132, USA tion of hardware cloth, epoxy, and plywood does not add much e-mail (BMG): [email protected] weight and retains the effectiveness of earlier designs, but renders The most primitive method used to capture turtles is by hand, the traps more durable (some of our traps have been in service for and a variety of hand capture methods appear in the literature (Cagle 10 years) and better able to handle numerous large snakes (Fig. 1950; Carpenter 1955; Marchand 1945). Non-baited traps, par- 2). Although we designed those traps to be placed on perimeter ticularly basking traps of various forms, have also been used in fences, they are versatile and can be placed at the end of a drift many turtle studies (Cagle 1950; Lagler 1943; Petokas and fence with leads (Fig. 2) or can be modified easily into a two- Alexander 1979; Robinson and Murphy 1975). The most popular ended funnel trap (by the addition of a second funnel) to be placed baited-trap method is the hoop net, originally described by Legler at the center of a fence. Compared to box designs, we believe our (1960), and later refined by others to suit their specific needs. Since mesh design is advantageous because it is light and see-through, Plummer (1979) reviewed collection methods for turtles, many but we think it is also more effective because it allows airflow. individuals have improved earlier trap designs and developed in- Airflow is likely a cue animals use to find an escape hole along a novative capture techniques (e.g. Kuchling 2003; Sharath and fence. If one makes many traps, the cost will be < US $20 per trap Hegde 2003). Here we describe a novel technique that uses baited because the materials can be purchased in large quantities (e.g., deep-water crawfish nets to capture carnivorous or omnivorous full plywood sheets, 30.5 m (100 feet) rolls of hardware cloth and turtles. We include some preliminary data using this technique window screening). In conclusion, the traps can be used to catch a and discuss the potential advantages and disadvantages of these variety of terrestrial herpetofauna in numerous environments. nets over traditional hoop nets. Two dozen custom-made deep-water crawfish nets were pur- Acknowledgments.—Funding was provided by the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Natu- chased for US $75/dozen from a private dealer in Chalmette, Loui- ral Resources, and Parks Canada. We are grateful to R. Reed for his com- siana. Deep-water crawfish nets were constructed from a 50.8 cm ments on our manuscript. diameter stainless steel ring (4.8 mm diameter) to which 16 mm black-dipped mesh was attached loosely to form a pocket (Fig. 1). LITERATURE CITED Three 30.5 cm ropes were attached to the steel ring at equal dis- tances from each other and were tied together at the other end to ALFORD, R. A., AND S. J. RICHARDS. 1999. Global amphibian declines: a form a knot. A 5.1 cm diameter, 1.9 cm thick cork was attached problem in applied ecology. An. Rev. Ecol. Syst. 30:133-165. above the knot followed by another knot to keep this cork in place. BLOUIN-DEMERS, G., K. A. PRIOR, AND P. J. WEATHERHEAD. 2000. Patterns When placed in water, the cork suspended the three ropes above of variation in spring emergence by black rat snakes (Elaphe obsoleta obsoleta). Herpetologica 56:175-188. the mesh and minimized interference caused by turtles attempting to feed. A larger, second cork (5.7 cm in diameter and 3.8 cm

Herpetological Review 37(2), 2006 185 with the substrate to reduce the chance of turtles, especially mud and musk turtles, from feeding beneath the net. To accomplish this, the aluminum pole was used to clear vegetation or debris from the water. After positioning the net, the larger cork was placed to one side of the net in a position to be easily grabbed with the rods of the aluminum pole. If the habitat allowed, the larger cork was placed out of the water on the bank or vegetation. Only in deep water was the larger cork ever directly above the net. When using the pole to check nets, care was taken to minimize disturbance to the water surface, as this alarmed feeding turtles. To check nets, the larger cork was grasped between the rods, and then the cork was pulled straight up, and over to shore with one quick, fluid motion. Some turtles attempted to crawl or swim out of the net as it was picked up. Thus, checking nets was most effi- ciently accomplished with two individuals: one individual picked up the net and the second individual used a dipnet to catch any turtles that fell out of the net as it was being moved to land. Larger turtles (e.g., Chelydra serpentina) did not always completely en- ter the net while feeding. However, as the net was being lifted from the water, larger turtles often had their jaws firmly attached to the bait and could be lifted, albeit temporarily, from the water. FIG. 1. Illustration of deep-water crawfish net. If a second person was present with the dipnet ready, the turtle could be netted. The frequency of checking nets depended on a variety of envi- thick) was positioned 91.4 cm above the smaller, first cork. The ronmental variables, including habitat type, weather, water depth, larger cork floated on the water surface and was used as a "catch" water clarity and turtle behavior. Initially, nets should be set for at to extract the net from the water with a hollow aluminum pole least fifteen minutes before checking to allow the scent of the bait (3.8 cm diameter, 195.6 cm long). Two stainless steel rods (6.4 to spread and attract turtles. However, because the scent was gen- mm in diameter) were attached on opposite sides of the same end erally detected quickly in smaller lentic waters, nets needed to be of the aluminum pole (Fig. 2). The rods were 30.5 cm long, but checked every ten minutes. If feeding activity was low, more time extended only 15.2 cm beyond the pole. For added strength the between checking nets was required. In clear, lotic waters turtles remaining length of the rods was placed inside the aluminum pole could be observed feeding. Thus, to avoid disturbing feeding turtles, and secured tightly by hammering flat the end of the pole. Both stealth was required in approaching and picking up the net. As rods were slightly curved to prevent the large cork from falling off turtles fed on the bait, oils and lipids rose and the presence of oil as the net is pulled from the water. The distance between the rods or small pieces of chicken fat at the water surface usually indi- was 2.5 cm at the point they contacted the pole, but increased to cated feeding. Typically, the more oils at the surface, the larger the 3.5 cm toward their end. The greater distance at the end of the turtle (or more than one turtle) that was feeding. Also, larger turtles rods facilitated grabbing the large cork, and as the net was pulled sometimes caused the larger cork at the surface to move while from the water, the cork slid down to the base of the rods where feeding in the net. they contacted the pole. The point where the rods contacted the pole was the strongest part of the pole, and the strain of picking up nets rested on this position, not on the end or middle of the rods. Each net was baited with chicken backs or leg quarter portions purchased from a local grocery. Each net was equipped with a center string for bait attachment, but to achieve longer bait life, a standard shower curtain clip was attached to the center of each net. The thickest part of the chicken back was pushed through the open clip, snaking the clip through the thin bone as many times as possible for a firm attachment, and then the clip was snapped close. Large turtles can remove poorly secured bait, and straighten clips while feeding; therefore, extra clips and replacement bait were required for an entire day of trapping. Deep-water crawfish nets were set in waters ranging in depth from 15.2 cm to 121.9 cm. Spacing between nets was variable and depended primarily on depth and clarity of the water body. In murky, lentic waters (e.g., canals, ponds, sloughs, etc.) nets were Fib. 2. Illustration of terminal end of aluminum pole showing position positioned close together. In clear, lotic waters (e.g., rivers, streams, of steel rods used to extract deep-water crawfish nets from water via the etc.) the distance between nets was increased. The nets rested flush large cork.

186 Herpetological Review 37(2), 2006 This technique has captured seven turtle species in several dif- Acknowledgments.-For field assistance we thank E. L. Young, V. A. ferent aquatic habitats, including drainage canals in St. Bernard Cobb, G. R. Wyckoff, T. Glorioso, and T. Niemiller. We are grateful to Parish, Louisiana (Kinosternon subrubrum, Sternotherus odoratus the University of Tennessee at Martin for housing at Reelfoot Lake. For and Trachemys scripta), floodplain ponds and the main channel comments on previous versions of this manuscript we thank B. T. Miller and an anonymous reviewer. For assistance with making figures suitable (S. odoratus, of the Stones River in Rutherford County, Tennessee for publication we thank M. Filopoulos. Funding for this research was T. scripta, Apalone spinifera, C. serpentina, and Graptemys supported in part by the Tennessee Wildlife Resources Agency (TWRA), geographica), and a roadside slough adjacent to Reelfoot Lake in National Park Service (NPS), and the Department of Biology, Middle Lake County, Tennessee (K. subrubrum, S. odoratus, T scripta, Tennessee State University (MTSU). Research was conducted under C. serpentina, and Chrysemys picta). At the roadside slough TWRA Scientific Collection Permit No. 192 and No. 1798, NPS Scien- (36°21.150'N, 89°24.920'W), 16 deep-water crawfish nets were tific Research and Collecting Permit No. STRI-2003-SCI-0004, and TDEC used on 25 September 2004 for eight hours (0900-1700 h). All 16 Scientific Collecting Permit #2005-001. Animals were treated ethically nets were set from the shore in a straight (ca. 190 m) stretch of the according to standards set forth in MTSU IACUC Protocol #04-008. slough. The total catch was 125 turtles with the following counts: LITERATURE CITED 1 1 1 S. odoratus, 8 C. picta, 5 T scripta, and 1 C. serpentina. The largest turtle captured using this technique was a C. BARKO, V. A., J. T. BRIGGLER, AND D. E. OSTENDORF. 2004. Passive fishing serpentina with a plastron length of 234 mm and a mass of 10.25 techniques: A cause of turtle mortality in the Mississippi River. J. Wildl. kg. This large turtle was captured with the aid of a dipnet, as it was Manage. 68:1145-1150. not entirely within the net when it was pulled from the water. CAGLE, F. R. 1950. The life history of the slider turtle, Pseudemys scripta Chelydra serpentina exceeding 10 kg fed in the nets, but managed troostii (Holbrook). Ecol. Monogr. 20:31-54. to escape during net retrieval. If the diameter of the ring for the CARPENTER, C. C. 1955. Sounding turtles: A field locating technique. nets were increased, this might increase chances of capturing these Herpetologica 11:120. larger snapping turtles. Trachemys scripta ranging in size from 42 DODD, C. K., JR. 1989. Population structure and biomass of Sternotherus odoratus (Testudines: Kinosternidae) in a northern Alabama lake. to 235 mm plastron length and 19.5 to 2575 g have been captured Brimleyana (15):47-56. using deep-water crawfish nets. The smallest turtle captured was KUCHLING, G. 2003. A new underwater trap for catching turtles. Herpetol. a hatchling S. odoratus with a plastron length of 16.6 mm having Rev. 34:126-128. a mass of 3.2 g. Therefore, these nets are suitable for capturing LAGLER, K. F. 1943. Methods of collecting freshwater turtles. Copeia nearly all size classes of carnivorous/omnivorous aquatic and semi- 1943:21-25. aquatic turtles. They are especially adept at capturing S. odoratus. LEGLER, J. M. 1960. A simple and inexpensive device for trapping aquatic These nets have a number of advantages over traditional hoop turtles. Proc. Utah Acad. Sci. 37:63-66. nets: 1) With this active method of catching turtles exact times of MARCHAND, L. J. 1945. Water goggling: A new method for the study of feeding can be ascertained, which could not be done with any pre- turtles. Copeia 1945:37-40. PETOKAS, P. J., AND M. M. ALEXANDER. 1979. A new trap for basking turtles. cision with hoop nets. 2) They are less expensive and less bulky Herpetol. Rev. 10:90. than hoop nets. In general, two dozen deep-water crawfish nets PLUMMER, M. V. 1979. Collecting and marking. In M. Harless and H. takes up less space than a traditional hoop net. 3) Turtles can be Morlock (eds.), Turtles: Perspective and Research, pp. 45-60. John captured in extremely shallow waters with these nets. 4) With hoop Wiley & Sons, New York, New York. nets, there have been reports of turtle injury or mortality resulting ROBINSON, K. M., AND G. G. MURPHY. 1975. A new method for trapping from prolonged periods within the net (e.g., Barko et al. 2004; softshell turtles. Herpetol. Rev. 6:111. Dodd 1989). The likelihood of injury using deep-water crawfish SHARATH, B. K., AND S. N. HEGDE. 2003. Two new traps for sampling the nets is significantly reduced because turtles are not 'trapped', and black pond turtle (Melanochelys trijuga) in the tropical rainforests of nets are checked frequently. 5) Lastly, unlike hoop nets, there is the Western Ghats (India). Herpetol. Rev. 34:33-34. little chance of theft or sabotage to deep-water crawfish nets that you are actively checking from the shore. Hoop nets do have some advantages over deep-water crawfish nets. Because hoop nets only have to be baited, set and checked every so often, the time required to sample in this manner is sig- nificantly less than sampling using deep-water crawfish nets. The actual trapping of hoop nets is done passively as opposed to the active method of using deep-water crawfish nets where the inves- tigator must be present. Hoop nets are advantageous in situations where trapping must be done from a boat. We suggest that deep- water crawfish nets will not work well in these situations because the surface disturbance created by a moving boat would scare feed- ing turtles out of the net before it could be checked. Also, hoop nets are useful in trapping turtles that feed at night. By compari- son, deep-water crawfish nets are more difficult to use at night. However, we increased our trapping success at night by wearing headlamps and affixing reflective tape to the large cork and rods of the aluminum pole.

Herpetological Review 37(2), 2006 187 and growth as our long-term responses because these variables Herpetological Review, 2006, 37(2), 188-191. 0 2006 by Society for the Study of Amphibians and Reptiles provide an indication of the general health of an amphibian and relate to demographic processes. Fluorescent Powder Pigments as a Harmless Materials and Methods.—Wood Frog egg masses were collected Tracking Method for Ambystomatids and Ranids from the Daniel Boone Conservation Area in Warren County, Mis- souri, USA, on 6 March 2004. After hatching, tadpoles were reared until metamorphosis in outdoor 1000-liter cattle tank mesocosms TRACY A. G. RITTENHOUSE* TIMOTHY T. ALTNETHER stocked with 1 kg leaf litter and zooplankton inoculum. Larval and Spotted Salamanders were collected (using dip nets) from the RAYMOND D. SEMLITSCH Baskett Wildlife Research Area in Boone County, Missouri, USA, Division of Biological Sciences, University of Missouri on 25 August 2004 and maintained in aquaria with aerated pond Columbia, Missouri 65211, USA water until metamorphosis. Newly metamorphosed frogs and sala- * Corresponding author e-mail: [email protected] manders were housed in aquaria with moist sphagnum moss at the University of Missouri and fed crickets ad libitum. Many amphibian species require both aquatic and terrestrial Two long-term experiments tested for differences in growth and habitats to fulfill their biphasic life cycle. Research often focuses survival of Wood Frogs and Spotted Salamanders covered and not on processes and life stages occurring in aquatic habitats because covered with powder. For the experiment on frogs, 70 individuals the high concentration of animals facilitates the logistics of re- were randomly assigned to six treatment groups based on powder search and strong density dependence in larvae suggests that popu- color on 6 June 2004: blue (N = 10), green (N = 10), orange (N = lations may be regulated by this life stage (Wilbur 1980). How- 10), yellow (N = 10), red (N = 10), and a no powder control (N = ever, adult amphibians use extensive amounts of terrestrial habi- 20). For the experiment on salamanders, 20 individuals were ran- tat (Semlitsch and Bodie 2003), populations worldwide are de- domly assigned to two treatment groups on 20 September 2004: clining due to habitat loss (Stuart et al. 2004), and new evidence red (N = 10) and a no powder control (N = 10). Treatment con- suggests that the juvenile or adult life stages may be important in sisted of dipping each individual in fluorescent powder pigments regulating populations (Biek et al. 2002). Efforts to study amphib- (Radiant Color, Richmond, California, USA) until completely ians in terrestrial habitats have increased greatly in the last de- covered. Control animals were handled in a similar manner, ex- cade, but limitations in finding, capturing or tracking animals away cept they were not dipped in powder. Animals were randomly as- from breeding sites still restrict our ability to answer basic eco- signed to an individual 17 x 12 x 9 cm plastic container that con- logical questions in terrestrial habitats. tained moist sphagnum moss with a fiberglass window screen lid. Fluorescent powdered pigments (hereafter referred to as pow- All animals were fed approximately 18% of their body weight in der) have been used to track a variety of organisms including small small crickets each week, split between two feedings. Every two mammals, reptiles and larval amphibians (Blankenship and Bryan weeks for a six-week period all animals were weighed and the 1990; Fellers and Drost 1989; Ireland 1973; Lemen and Freeman powder treatment was re-applied. 1985). More recently, powder has been used to track amphibians Two short-term experiments tested for differences in the rate of in terrestrial habitats (Birchfield and Deters 2005). The primary water loss between animals covered and not covered with pow- assumption of all tracking studies is that the tracking method does der. Similar procedures were followed for both the wood frog ex- not affect the animal (White and Garrott 1990; Millspaugh and periment on 12 August 2004 and the spotted salamander experi- Marzluff 2001). Experimentally testing this assumption is an im- ment on 9 November 2004. Animals were placed in a plastic con- portant step to validate this technique. While reports of negative tainer containing 0.5 cm of carbon-filtered water for approximately effects of powder tracking are rare, the inhalation of powder was 12 hrs prior to the beginning of the experiment to ensure all ani- reported to cause moderate levels of histiocytic pneumonia in deer mals were fully hydrated. The dehydration chamber consisted of a mice, Peromyscus maniculatus (Stapp et al. 1994), and powder square chamber (5 x 5 x 5 cm) constructed of metal window screen can persist in the environment for long periods of time (Halfpenny (similar to Pough et al. 1983) that was suspended, exposing all 1992). Studies examining the effects of powder on amphibians sides to air and did not prevent animals from using water conserv- are limited (but see Berger 2000; Eggert 2002). ing postures. Animals were assigned to a powder treatment or con- We experimentally tested both short-term (i.e., powder present trol group according to the treatment that individual received dur- on the skin) and long-term effects of powder on both recently ing the long-term experiments, thus 20 frogs (i.e., N c.„„,,, = 10 and metamorphosed Wood Frogs, Rana sylvatica, and Spotted Sala- Med = 10) and 18 salamanders (i.e. N.., = 10 and Nred = 8) were manders, Ambystoma maculatum. We chose the rate of water loss tested. Using the same animals in both the short-term and long- as the short-term response because water regulation is a critical term experiments did not bias our results because the data from process for amphibians in terrestrial habitats and because amphib- the experiments were analyzed separately and because any poten- ian skin affords virtually no protection from desiccation (Ray 1958; tial carryover effects from the long-term experiments should in- Thorson 1955). The large surface area to volume ratio in recently crease the likelihood of detecting an effect in the short-term ex- metamorphosed juveniles causes water loss to be a greater threat periments. Each chamber was weighed, an animal was randomly to juveniles than adults (Thorson 1955). In addition, the small assigned to the chamber, and the combination of the animal and particle size of the powder coating the skin may alter the flow of chamber was weighed every 30 minutes for 120 minutes until ani- water across the skin and thus presents a possible mechanism for mals lost approximately 15% of their body mass. Thus animals how the powder could affect an amphibian. We chose survival were not exposed to lethal dehydration levels: 30-35% for ranids

188 Herpetological Review 37(2), 2006 (Thorson and Svihla 1943); and 36-40% for ambystomatids (Pough and Wilson 1970; Ray 1958). control Changes in mass over six weeks for the A blue powder long-term experiments and over 120 min- 0.9 - green powder utes for the short-term experiments were orange powder analyzed using repeated measures analysis yellow powder of variance. Only animals without missing - red powder observations (i.e., individuals that survived 0.8 - the entire experiment) were included in the analysis of mass. Analysis of variance was used to test for the effects of the powder treatment on survival, with number of days 0.7 - alive as the response variable. All weights were obtained using a Mettler AT261 Delta Range electronic balance with readability 0.6 -

of 0.01mg. SD) Results.—Growth between animals cov- ± A ered and not covered with powder did not differ for either Wood Frogs (F = 0.19, d.f. an = 5,57, P = 0.97; Fig. la) or Spotted Sala- 0.5 manders (F = 0.24, d.f. = 1,17, P = 0.63; Me

Figure 1b). A significant increase in mass (g, 2- occurred throughout the six weeks for both

Wood Frogs (F = 354.77, d.f. = 3,55, P < Mass 0.0001) and Spotted Salamanders (F = 213.00, d.f. = 3,15, P < 0.0001). No interac- dy 1.8 - tions between the powder treatments and Bo time occurred (all P 0.40). The number of days alive did not differ between frogs cov- ered and not covered with powder (F = 0.58, 1.6 - d.f. = 5,64, P = 0.71), but seven frogs died throughout the course of the long-term ex- periment. Survival was 100% in the long- term salamander experiment, but one es- 1.4 - caped and one from the red treatment died / after the completion of the long-term experi- / ment but prior to the short-term experiment. 1.2 - / We believe the mortality occurred when animals were not feeding readily, because B these individuals were the smallest at the initiation of the experiments. 1 Water loss between the control and pow- der treatments did not differ for either Wood 0 14 28 42 Frogs (F = 0.34, d.f. = 1,18, P = 0.57; Fig. 2a) or Spotted Salamanders (F= 0.49, d.f. = Days 1,16, P = 0.49; Fig. 2b). Continuing the ex- FIG. 1. Mean growth for each powder treatment during the long-term experiments for Wood periment until all animals had lost approxi- Frogs (A) and Spotted Salamanders (B) at two week time intervals. mately 15% of their body mass produced a significant decrease in mass for both the frogs (F = 590.04, d.f. = 4,15, P < 0.0001) and the salamanders (F suggest that being covered with powder is similar to being cov- = 442.29, d.f. = 4,13, P < 0.0001). No interactions between pow- ered with other natural items, such as soil or organic debris, and der treatments and time occurred (all P 0.34). Survival was 100% conclude that powder is a harmless method for tracking in both short-term experiments. ambystomatids and ranids in terrestrial habitats. Discussion.—We did not detect any short-term or long-term ef- All experiments provided conditions that may cause the pow- fects of powder on either Wood Frogs or Spotted Salamanders. der to be more stressful than animals would experience when pow- Growth and survival over a six-week period, as well as rates of der is used to track amphibians in the field. First, animals in the water loss, were unaffected by being covered with powder. We laboratory had limited opportunity to remove the powder by rub-

Herpetological Review 37(2), 2006 189 1.2 - by transmitter implantation). Tracking amphibians with powder - red often provides the shortest movement paths of all the tracking methods; however, several benefits can make powder the preferred tracking method in many instances. Powder tracking results in a 0.8 - detailed description of the movement path (e.g., Birchfield 2002; Eggert 2002). Powder can be used on juveniles or species too small 0.6 - for other tracking devices and is relatively inexpensive (e.g., US $12 per one-pound can). However, a possible side effect to am-

0.4 - phibians is the potential increase in visibility to predators that use 0.2 - color to locate prey. Although the optimum tracking method will ± SD) A n vary based on the research objectives of a study, tracking amphib- 0 ians with powder is an underutilized tracking technique that does

Mea not appear to detrimentally affect growth, water regulation or sur-

(g, 2 vival in the laboratory of ambystomatids or ranids. Therefore, this technique might be particularly useful when studying rare or en-

Mass 1.6 - dangered species. dy

Bo 1.2 - Acknowledgments. -We thank E. Harper for raising the Wood Frogs to metamorphosis. J. Crawford provided thoughtful comments on the manu- script. Animals were captured under Missouri Department of Conserva- 0.8 - tion Wildlife Collector's permits 12220 and 12227 and maintained under University of Missouri Animal Care and Use protocol 3368. Funding was 0.4 - B provided by NSF grant DEB 0239943 to R. Semlitsch. LITERATURE CITED 0 0 30 60 90 120 BARTELT, P. E., C. R. PETERSON, AND R. E. KLAVER. 2004. Sexual differ- Minutes ences in the post-breeding movements and habitats selected by west- ern toads (Bufo boreas) in southeastern Idaho. Herpetologica 60:455- FIG. 2. Mean water loss for each powder treatment during the short- 467. term experiments for wood frogs (A) and spotted salamanders (B) at 30 BERGER, L. 2000. Estimation of methods of individual marking of anuran min time intervals. amphibians. Prezeglad Zoologiczny 44:23-28. BIEK, R., W. C. FUNK, B. A. MAXELL, AND S. Mills. 2002. What is missing bing against vegetation or hopping around. This was especially in amphibian decline research?: Insights from ecological sensitivity true in the dehydration experiments where small enclosures re- analysis. Cons. Biol. 16:728-734. BIRCHFIELD, G. L. 2002. Adult green frog (Rana clamitans) movement stricted hopping movements and substrate was not provided. Ani- behavior and terrestrial habitat use in fragmented landscapes in central mals in these experiments remained completely covered with pow- Missouri. Ph.D. Thesis, University of Missouri, Columbia, Missouri, der for the entire 120 minutes. Although we did not observe any USA. behavior that suggested animals were purposely attempting to re- , AND J. E. DETERS. 2005. Movement paths of displaced northern move the powder, animals in the long-term experiments lost the green frogs (Rana clamitans melanota). Southwest. Nat. 4:63-76. powder quickly, with powder visible on approximately 52% of BLANKENSHIP, E. L., AND T. W. BRYAN. 1990. A method for tracking tor- the animals at 24 h after powder application and no powder vis- toises using fluorescent powder. Herpetol. Rev. 21:88-89. ible on any of the animals at 3 days after powder application. Sec- EGGERT, C. 2002. Use of fluorescent pigments and implantable transmit- ond, animals in the long-term experiments were exposed to the ters to track a fossorial toad (Pelobates fuscus). Herpetol. J. 12:69-74. FELLERS, G. M., AND C. A. DROST. 1989. Fluorescent powder-a method powder on three occasions. When powder is applied in the field, for tracking reptiles. Herpetol. Rev. 20:91-92. animals are often only covered with powder once, because the HALFPENNY, J. C. 1992. Environmental impacts of powder tracking using individual is not recovered after being released. We found no evi- fluorescent pigments. J. Mammal. 73:680-682. dence that repeated exposure to the powder is harmful to amphib- IRELAND, P. H. 1973. Marking larval salamanders with fluorescent pig- ians. ments. Southwest. Nat. 18:252-253. Three tracking methods are primarily used for directly follow- LEMEN, C. A., AND P. W. FREEMAN. 1985. Tracking mammals with fluores- ing amphibians in terrestrial habitats: radio-telemetry, thread-trail- cent pigments: a new technique. J. Mammal. 66:134-136. ing, and fluorescent powder pigments. Each method has advan- MILLSPAUGH, J. J., AND J. M. MARZLUFF. 2001. Radio Tracking and Animal tages and disadvantages. For example, although radio-telemetry Populations. Academic Press, New York. 474 pp. POUGH, F H., T. L. TAIGEN, M. M. STEWART, AND P. E BRUSSARD. 1983. allows a researcher to track an individual for the longest time pe- Behavioral modification of evaporative water loss by a Puerto Rican riod (e.g., 1-4 months depending on transmitter size) and longest frog. Ecology 64:244-252. distances (e.g., Bartelt et al. [2004] tracked Bufo boreas > 200 m), , AND R. E. WILSON. 1970. Natural daily temperature stress, dehy- the cost of radio-telemetry is the greatest (US $150 per transmitter dration, and acclimation in juvenile Ambystoma maculatum (Shaw) plus additional costs for receiving equipment) and risk to the ani- (Amphibia: Caudata). Physiol. Zool. 43:194-205. mal can be the greatest (see Rittenhouse 2002 for mortality caused RAY, C. 1958. Vital limits and rates of desiccation in salamanders. Ecol- ogy 39:75-83.

190 Herpetological Review 37(2), 2006 RITTENHOUSE, T. A. G. 2002. Spotted salamander migration at a pond lo- tuations in lighting conditions can cause errors and alter the radi- cated on a forest-grassland edge. Master of Arts, University of Mis- ance or reflectance output given by a spectrometer (Endler 1990, souri, Columbia, Missouri. 1993). Thus, a method that eliminates or greatly reduces inconsis- SEMLITSCH, R. D., AND J. R. BODIE. 2003. Biological criteria for buffer tencies in lighting would provide far more consistent and repeat- zones around wetlands and riparian habitats. Cons. Biol. 17:1219-1228. able measurement of reflectance or radiance. In particular, the STAPP, P., J. K. YOUNG, S. VANDEWOUDE, AND B. VAN HORNE. 1994. An evaluation of the pathological effects of fluorescent powder on deer exclusion of ambient light, which can change over short time pe- mice (Peromyscus maniculatus). J. Mammal. 75:704-709. riods because of movement of clouds or overhead vegetation STUART, S. N., J. S. CHANSON, N. A. Cox, B. E. YOUNG, A. S. L. RODRIGUES, (Endler 1993), would increase the constancy of reflectance spec- D. L. FISCHMAN, AND R. W. WALLER. 2004. Status and trends of amphib- tra. Yet such a method has limitations when used in the field, be- ian declines and extinctions worldwide. Science 306:183-186. cause of the difficulty of removing all sources of ambient light. THORSON, T. B. 1955. The relationship of water economy to terrestrialism Until now these limitations have meant that study subjects have in amphibians. Ecology 36:100-116. been returned to the laboratory to take controlled reflectance read- , AND A. SVIHLA. 1943. Correlation of the habitats of amphibians ings in a darkened room (Macedonia et al. 2002; Stuart-Fox et al. with their ability to survive the loss of body water. Ecology 24:374- 2003, 2004). However, removing lizards from their natural habi- 381. tat may result in stress responses, which could lead to skin-color WHITE, G. C., AND R. A. GARRarr. 1990. Analysis of Wildlife Radio-track- ing Data. Academic Press, San Diego. 383 pp. changes, as reported in species that have the ability to rapidly WILBUR, H. M. 1980. Complex life cycles. Ann. Rev. Ecol. Syst. 11:67— lighten or darken their skin color, such as some agamids (Chris- 93. tian et al. 1996) and iguanians (e.g., Cooper and Greenberg 1992). When investigating background-color matching, such color changes may not be indicative of the lizard's typical body color. We have designed and tested an opaque probe cover for use with a portable spectrometer that allows accurate and controlled Herpetological Review, 2006, 37(2), 191-194. measurement of reflectance in the field. We tested the reliability (0 2006 by Society for the Study of Amphibians and Reptiles of this new method during a study of background-color matching A New Technique for Measuring Body Color of in the painted dragon, Ctenophorus pictus. In this article we de- Lizards in the Field scribe an effective method for measuring reflectance of virtually any species of lizard in the field with the aid of this probe cover. In fact, the methods described would have applicability across a REBECCA J. ROSE wide range of animal taxa. Our methods are inexpensive, easily Department of Zoology, University of Melbourne Parkville, Victoria 3052, Australia constructed, and portable. and Cover for Optical Fiber Probe.—Measures of reflectance were Department of Sciences, Museum Victoria, GPO Box 666 taken using an Ocean Optics USB2000 Miniature Fiber Optic Melbourne, Victoria 3001, Australia Spectrometer® (Dunedin, Florida) and an illumination source was e-mail: [email protected] provided by a PX-2 Pulsed Xenon Lamp® (Dunedin, Florida), con- and nected to the spectrometer by a standard reflection probe (200 p.m JANE MELVILLE diameter). The spectrometer and light source were then connected Department of Sciences, Museum Victoria, GPO Box 666 to a laptop computer via a USB cable for reflectance calculations Melbourne, Victoria 3001, Australia e-mail: [email protected] using the Ocean Optics software package, OOIBase32. We designed an opaque cover that attaches to the optical fiber Measurement of color in reptiles can play a fundamental role in probe, ensuring that only the light from the xenon lamp illumi- research areas as diverse as behavior, physiology, evolution, and nates the target area. This cover was constructed from a large plastic ecology. For example, studies incorporating color have investi- drinking straw that fit snugly over the probe (Fig. 1). The straw gated the behavioral role of dewlap color in Anolis lizards was wrapped in layers of black duct tape to block out ambient (Macedonia et al. 2003; Thorpe and Stenson 2003), thermoregu- light. One end of the straw was then cut to make a 45 ° angle at a lation in desert reptiles (Norris 1967), sexual selection in chuck- distance of 1 cm from the end of the probe to the surface sampled wallas (Kwiatkowski and Sullivan 2002), and predator evasion in (following Endler 1990). Once the probe was cut to size, several agamid lizards (Stuart-Fox et al. 2004). These studies rely on the layers of duct tape were placed around the cut end of the probe to ability to accurately quantify body color. create a base (10 x 10 cm), which further reduced light from en- There are a number of small portable spectrometers available tering the receiving end of the probe. This base can be made to that measure reflectance or radiance. Previous studies using spec- any size and/or shape to suit different-sized lizards. The resulting trometers to measure the color of lizards and/or their backgrounds probe cover is flexible and after being placed on a lizard can be in the field typically used sunlight as the source of illumination peeled backwards to ensure the probe is positioned in the desired (Macedonia et al. 2002, 2003). This approach can cause problems location. The lizard is restrained by wrapping the base of the probe if the measurements are not taken under similar and constant con- around the body until the measurement was made but we also rec- ditions, as weather conditions, nearby vegetation, different mi- ommend restraining the lizard while measurements are being taken crohabitats, seasons, and the time of day can result in drastically (e.g., Rose et al. 2006). Additionally, a transparent probe cover different radiance spectra of the animal or background being mea- was constructed to test the accuracy of the opaque-probe cover sured (Endler 1990, 1993). Consequently, moment-to-moment fluc- using the same methods described for the opaque cover, the only

Herpetological Review 37(2), 2006 191 difference being that clear tape was used in- stead of black duct tape to create a transpar- D-G =A- F= 1.0cm ent rather than opaque cover. B-C=E- F=0.5 cm C-E= B- F=A- B= 0.7 cm All measurements were expressed relative C - D = 0.35 cm to a white reflectance standard (> 95-98% circumference = 3.5 cm reflectivity). Lizards were placed on a black backing during the readings to ensure that only the reflectance of the lizard was being measured. The probe cover, where it meets the surface being measured, has an area of Optical fibre probe 0.55 cm2, meaning that the surface quanti- fied would need to be at least this size to ensure all ambient light was eliminated. The area of color being measured with the opaque surface measured cover is equivalent to the diameter of the optical fiber aperture used (200 gm), with the area of light leaving the probe equal to FIG. 1. Design of the cover for the optical fiber probe. 0.31 cm2. Testing the Efficiency of the Opaque Probe Cover.—To deter- parent probe cover, and opaque probe cover) for four color stan- mine whether our opaque probe cover provided an improvement dards (blue, green, red, and yellow) across three lighting environ- over measuring radiance in ambient light, we conducted an ex- ments (darkened room, outdoors full-light, and outdoors shaded). periment using color standards. We tested the opaque cover by The eigenvalues and percentage of explained variance were as measuring color standards (blue, green, red, and yellow) in a dark- follows: PC1 (17845.72, 69.43%); PC2 (5717.55, 22.24%); PC3 ened room, outdoors in full light, and outdoors under the shade of (1936.52, 7.53%). We compared the three measurement methods a tree. Three methods (no probe cover, transparent probe cover, in each of the light conditions for each PC separately, using paired and opaque probe cover) were tested in each light environment to t-tests. In outdoor full light we found significant differences be- determine if there were significant differences between spectral tween using the opaque probe cover and no probe cover for PC2 readings among the three methods. (t3 = -3.469, P = 0.040) and PC3 (t3 = -7.950, P = 0.004), and In obtaining the spectral data, the parameters for each method between the opaque probe cover and the transparent probe cover could not be kept constant. The integration times for the spectral for PC2 (t3 = -3.856, P = 0.031) and PC3 (t3 = -10.935, P = 0.002). readings had to be adjusted (Table 1) to ensure that the reference In outdoor shaded light we found significant differences between calibration was not being saturated. As a result, we standardized using the opaque probe cover and no probe cover for PC2 (t 3 = - the maximum height of the peak for the standard in scope mode to 3.208, P = 0.049) and PC3 (t3 = -7.027, P = 0.006) and between approximately 3500 counts and used the corresponding integra- the opaque probe cover and the transparent probe cover for PC2 tion time. (t3 = -4.711, P = 0.025) and PC3 (t3 = -4.288, P = 0.023). In the The opaque probe cover was highly consistent across all three shaded condition there was also a significant difference between light environments (Table 1), as it did not require the integration using no probe cover and using the transparent probe cover in time to be altered. The integration times for the opaque probe cover PC1 (t3 = 3.350; P = 0.044). There were no significant differences were also equivalent to the integration time needed without a probe among the three methods in a darkened room. cover in a darkened room. This suggests that the opaque cover can These results indicate that there are significant differences be- be used in a variety of light environments and still be equivalent tween using the opaque probe cover and using no probe cover or a to taking readings without a probe cover in a darkened room. clear probe cover across different lighting conditions, especially All spectral data presented throughout this study were analyzed in terms of PC2 and PC3. These differences are most likely to be from 320-720 nm. This range includes the spectrum visible to a result of changes in ambient light from the time when the white humans (400-700 nm) as well as some ultraviolet (UV) (below standard sample was recorded, and when the color standard was 400 nm) and infrared (above 700 nm) regions. This region was measured. This could be the result of changes in reflected radia- chosen for a study of the body color of Ctenophorus pictus be- tion (e.g., cloud cover, vegetation), or experimental error such as cause many animals see beyond the human spectrum, including subtle changes in the lizard's body position among readings. Nev- birds (Bennett and Cuthill 1994; Hart et al. 2000) and some liz- ertheless, the opaque probe cover performed most consistently ards (Fleishman et al. 1993). across a range of lighting conditions, regardless of whether the The raw reflectance data were grouped into 10-nm bins for sta- ambient light conditions were varied. tistical analysis, which reduces the problem of non-independence Testing the Opaque Probe Cover on a Study Species.—We tested of data points (Leal and Fleishman 2002; Macedonia et al. 2002; for dorsal background-color matching in two populations of Thorpe and Stenson 2003). We analyzed the reflectance data by Ctenophorus pictus. Five females were collected from the Little principal components analysis (PCA) to reduce the number of Desert National Park (36°34'02"S; 141 °20'55"E; white sand popu- variables for analysis (Endler 1990; Macedonia et al. 2003). Three lation) and five were from the Murray Sunset National Park principal components (PCs) were extracted from the PCA com- (36°34'46.5"S; 141°36'38"E; red sand population) in northwest- bining the three methods of data acquisition (no probe cover, trans- ern Victoria. Lizards were measured outdoors, under natural light

192 Herpetological Review 37(2), 2006 TABLE 1. Integration times (ms) needed in each light environment for field even without a support vehicle if researchers were willing to each probe cover to obtain 3500 counts in scope mode. transport a spotlight or car battery into the field. Our opaque probe cover provides an inexpensive easy to con- Integration Time (ms) struct method to measure reflectance across that should prove suit- able for a wide range of taxa. We have shown the advantages of Probe cover Inside Full light Tree shade measuring reflectance in the field. In addition, this method is ap- plicable to measuring the reflectance of any surface > 0.55 cm 2. In No cover 110 30 70 the past, optical fiber probe covers have been used to hold the Transparent cover 70 35 70 probe at a constant distance and 45 ° angle to the surface (Stuart- Opaque cover 110 110 110 Fox et al. 2004; Thorpe 2002; Thorpe and Stenson 2003). How- ever, these covers were not designed to block out ambient light from entering the surface being measured. Other studies have re- conditions, using the opaque cover and then these measurements lied on attaching a ruler to the end of the probe to maintain a con- were repeated indoors in a darkened room without use of the probe stant distance between the probe and the surface being measured cover, which is a more typical method of color measurement. To (Macedonia et al. 2002, 2003). Our probe cover is an improve- provide consistency in the tests, we used the same spectrometer ment on previous methods because, as well as providing constant settings across all measurements. distances between the probe and the surface of analysis, it also The lizards were warmed to 37.8°C immediately before reflec- blocks out ambient light, making it suitable for repeatable mea- tance was measured. This value was chosen as it lies within the surements of reflectance in the field. two published mean active body temperatures of this species (34.4°C, Melville and Schulte 2001; 39.0°C, Mayhew 1963). Four Acknowledgments.-We thank L. Revell for his assistance with the reflectance readings were taken for each lizard on the dorsal sur- spectrometer and analysis. We are grateful to J. Van Buskirk and J. Ng for face of the body: head, between the shoulders, mid-back, and at their advice and assistance. We are especially thankful for the valuable the base of the tail. These locations encompass the range of dorsal comments made by two anonymous reviewers. Permits for field work were provided by the Department of Sustainability and Environment color and pattern variance for this species. We measured the dor- Victoria (Permit No. 10002952), and the Department for Environment sal surface because we were interested in background-color match- and Heritage South Australia (Permit No. E24870). Funding for this study ing. Readings on the mid-back of the lizard were taken slightly off was provided by ARC Discovery Grant (DPO452082) and Australian center to avoid the black patterning along the middle of the back. Society of Herpetologists Student Research Grant. This research was ap- This was achieved by folding back the flexible opaque cover as it proved by The University of Melbourne Faculty of Science Animal Ex- was being placed on the back of the lizard to ensure the precise perimental Ethics Committee and The South Australian Wildlife Ethics locality of reflectance readings. Committee. A PCA reduced the data set to three axes, with eigenvalues and the percentage of explained variance as follows: PC1 (1658.77, LITERATURE CITED 88.29%); PC2 (202.52, 10.78%); PC3 (10.52, 0.56%). To test for BENNETT, A. T. D., AND I. C. CUTHILL. 1994. Ultraviolet vision in birds: differences between using the probe cover outside and measuring what is its function? Vision Res. 34:1471-1478. reflectance inside, we used a one-way repeated measures ANOVA CHRISTIAN, K. A., G. S. BEDFORD, AND S. T. SHANNAHAN. 1996. Solar ab- for each of PC1, PC2, and PC3. No significant difference was sorptance of some Australian lizards and its relationship to tempera- detected in using the probe cover outside in natural light, or taking ture. Aust. J. Zool. 44:59-67. reflectance measures in a darkened room without a probe cover COOPER, W. E. JR, AND N. GREENBERG. 1992. Reptilian coloration and be- for each PC score (PC1: F1 9 = 0.380; P = 0.553; PC2: F1 9 = havior. In C. Gans and D. Crews (eds.), Biology of the Reptilia: Physi- ology E. Hormones, Brain, and Behavior, pp. 299-400. Univ. Chicago 0.006; P = 0.940; PC3: FI 9 = 2.766; P = 0.131). A problem that we noted in taking the readings inside without the probe cover Press, Chicago, IL. ENDLER, J. A. 1990. On the measurement and classification of colour in was the difficulty in estimating the constant 1-cm distance and studies of animal colour patterns. Biol. J. Linn. Soc. 41:315-352. 45° angle desired for taking readings. 1993. The color of light in forests and its implications. Ecol. Next, we measured the dorsal reflectance of 112 Ctenophorus Monogr. 63:1-27. pictus in the field using the opaque cover. Because C. pictus are FLEISHMAN, L. J., E. R. LowE, AND M. LEAL. 1993. Ultraviolet vision in easily caught in these sandy deserts, we could measure their re- lizards. Nature 365:397. flectance immediately at the point of capture. We also measured HART, M. S., J. C. PARTRIDGE, AND I. C. CUTHILL. 2000. Visual pigments, the reflectance of the sand the lizard was on at the point of cap- oil droplets, ocular media and cone photoreceptor distribution in two ture, thereby permitting estimates of background-color matching species of passerine bird: the blue tit (Parus caeruleusL.) and the black- in situ. bird (Turdus merula L.). J. Comp. Physiol. A. 186:375-387. KWIATKOWSKI, M. A., AND B. K. SULLIVAN. 2002. Geographic variation in an ideal study species because they are Ctenophorus pictus is sexual selection among populations of an iguanid lizard, Sauromalus easily caught within proximity of our field vehicle. Consequently, obesus (= ater). Evolution 56:2039-2051. the light source could be powered by the vehicle's battery: a volt- LEAL, M., AND L. J. FLEISHMAN. 2002. Evidence for habitat partitioning age transformer was plugged into the car's cigarette lighter, and a base on adaptation to environmental light in a pair of sympatric lizard 25-m extension cord was attached to the transformer. This allowed species. Proc. Roy. Soc. Lond. B 269:351-359. us to measure the reflectance of lizards and their backgrounds up MACEDONIA, J. M., Y. BRANDT, AND D. L. CLARK. 2002. Sexual dichroma- to 25-m away from the vehicle. Our method could be used in the tism and differential conspicuousness in two populations of the com-

Herpetological Review 37(2), 2006 193 mon collared lizard (Crotaphytus collaris) from Utah and New Mexico, vent length (SVL). Our technique reduces the time spent process- USA. Biol. J. Linn. Soc. 77:67-85. ing individual animals and may, therefore, minimize handling , A. V. ECHTERNACHT, AND J. W. WALGARNEY. 2003. Color varia- stress. Our method is easily constructed, inexpensive, and por- tion, habitat light, and background contrast in Anolis carolinensis along table making it suitable for both laboratory and field studies. We a geographical transect in Florida. J. Herpetol. 37:467-478. used this restraining method successfully on three species of MAYHEW, W. W. 1963. Observations on captive Amphibolurus pictus an Australian agamid lizard. Herpetologica 19:81-88. agamid lizards (Painted Dragon, Ctenophorus pictus; Mountain MELVILLE, J., AND J. A. SCHULTE II. 2001. Correlates of active body tem- Dragon, Rankinia diemensis; and Bearded Dragon, Pogona peratures and microhabitat occupation in nine species of central Aus- vitticeps) and one species of gecko (Knob-tailed Gecko, Nephrurus tralian agamid lizards. Austral Ecol. 26:660-669. amyae). Specifically, we provide detailed descriptions of the meth- NORRIS, K. S. 1967. Color adaptation in desert reptiles and its thermal ods used to restrain Ctenophorus pictus in the field for morpho- relationships. In W. W. Milstead (ed.), Lizard Ecology: A Symposium, metric analysis using digital photography. pp. 162-229. Univ. Missouri Press, Columbia, Missouri. Restraining Tray.—When using digital photography to conduct ROSE, R. J., J. NG, AND J. MELVILLE. 2006. A technique for restraining morphometric analyses it is critical to maintain all lizards in the lizards for field and laboratory measurements. Herpetol. Rev. 37:194- same position for each image, and to provide an unobstructed view 195. of the appendages. With this in mind, we designed a restraining STUART-FOX, D. M., A. MOUSSALLI, N. J. MARSHALL, AND I. P. F. OWENS. 2003. Conspicuous males suffer higher predation risk: visual model- tray using single-sided Velcro with an adhesive backing to hold ing and experimental evidence from lizards. Anim. Behay. 66:541- the animal's body in place, and a plastic tray as the base. Strips of 550. the hooked side of Velcro were stuck onto the tray, and thin strips , A. MOUSSALLI, G. R. JOHNSTON, AND I. P. F. OWENS. 2004. Evolu- of the looped Velcro were cut and the adhesive backing covered in tion of color variation in dragon lizards: quantitative tests of the role of flexible cotton material to avoid dirt and sand sticking to it. Liz- crypsis and local adaptation. Evolution 58:1549-1559. ards were placed onto the Velcro base of the tray and positioned as THORPE, R. S. 2002. Analysis of color spectra in comparative evolution- needed. We restrained lizards with one strip of Velcro placed firmly ary studies: molecular phylogeny and habitat adaptation in the St. over the neck, plus a second strip just above the pelvic girdle (Fig. Vincent anole (Anolis trinitatis). Syst. Biol. 51:554-569. 1). In the case of juveniles or very small lizards, one strip of Velcro , AND A. G. STENSON. 2003. Phylogeny, paraphyly and ecological adaptation of the colour and pattern in the Anolis roquet complex on across the neck was adequate for restraint. We surrounded the base Martinique. Mol. Ecol. 12:117-132. of hooked Velcro with strips of looped Velcro, which allowed the claws of the lizards to grip onto the base, ensuring that the limbs remained in the desired position. Morphometric Analysis.—For the morphometric analysis of

Herpetological Review, 2006, 37(2), 194- 195. Ctenophorus pictus, we attached a clear plastic ruler to the re- 0 2006 by Society for the Study of Amphibians and Reptiles straining tray and a label was included in each image for identifi- A Technique for Restraining Lizards for cation purposes. We then took digital photographs of restrained Field and Laboratory Measurements individuals, ensuring that the ventral surface of the lizards was flat on the tray and that their body was straight (Fig. 1). At least one hind limb and one forelimb were physically extended on the REBECCA J. ROSE restraining tray to ensure accurate measurement of limb propor- JULIENNE NG tions from the digital image. One advantage of digital images is Department of Zoology, University of Melbourne Parkville, Victoria 3052, Australia that the photographs can be archived and accessed at any time for and analysis. Department of Sciences, Museum Victoria, GPO Box 666 Six measurements were taken for each lizard (N = 128): head Melbourne, Victoria 3001, Australia length, SVL, axilla-groin distance, forelimb length, hind limb e-mail: bec_rose5@hotmaiLcom length, and tail length. Lizards measured included juveniles and and adults of both sexes ranging from 31.2-77.9 mm SVL. All lizards JANE MELVILLE measured were successfully restrained using our device, without Department of Sciences, Museum Victoria, GPO Box 666 a single lizard escaping or being injured. Melbourne, Victoria 3001, Australia e-mail: [email protected] Morphological analyses from the photographs were performed using ImageJ (v. 1.32j) digital image analysis software (Rasband Numerous techniques have been used for restraining lizards 2004). We tested the accuracy of measuring a subset of the mor- including controlling by hand, anesthetizing (Nelson and Jayne phological characters by comparing the measurements made with 2001), and thermal cooling combined with the use of sticky tape the digital images and analysis software to those recorded with (Hoefer et al. 2003). Restraining devices are often designed for digital calipers. The measurements using ImageJ differed by 1.7% use with members of specific genera (Poulin and Ivanyi 2003) or (N = 20) from caliper measurements, which would represent a 0.9 for lizards of a particular size (Hoefer et al. 2003). Such methods mm difference in the measurement of a 50 mm SVL lizard. impose limitations on studies that seek to measure morphological The restraining tray proved ideal for the wide range of sizes of characteristics of live lizards representing a broad range of body agamid lizards in our study. Our success across these agamid spe- sizes. cies may be a result of the rough scales and well-defined neck in Here we describe an effective technique for restraining various these lizards, which facilitated holding them in place with the species of lizards that range in body size from 30-250 mm snout- Velcro strips. Hence, this method should be applicable to most

194 Herpetological Review 37(2), 2006 ZU Z1 LL .L5 Z4 Zb *Lb *4 Zii 8 9 10 11 12 13 14 15 16 17 16 19 2 3 4 5 6 7 NEW IN TAURUS 9 300 FS CJ Ott 00Z 061 Ott Ott 09t 0Sl Ott 0E1 Olt Ott Ott 06 08 OL 09 OS 017 OE OZ Ot 09l 01Z 091 OSZ OCZ OCZ Olt

Ctenophorus niclus

Specimen Number:

Date: LP') ' D°1t Location: "Troo-A-

FIG. 1. Digital image of an adult male Ctenophorus pictus positioned on the restraining tray.

lizard taxa with rough scales and defined necks, such as agamids, NELSON, F. E., AND B. C. JAYNE. 2001. The effects of speed on the in vivo iguanids, cordylids, some lacertids, and many geckos. In fact, we activity and length of a limb muscle during the locomotion of the successfully used this restraining method on the gecko Nephrurus iguanian lizard Dipsosaurus dorsalis. J. Exp. Biol. 204:3507-3522. amyae without any damage to its delicate "knob-tail." This method POULIN, S., AND C. S. IVANYI. 2003. A technique for manual restraint of ensures that lizards are only held for a short period of time, there- helodermatid lizards. Herpetol. Rev. 34:43. fore reducing prolonged stress, which can result in tail autotomy. RASBAND, W. 2004. ImageJ 1.32j. National Institutes of Health, USA. Available on the web at: http://rsb.info.nih.gov/ij/. Our technique is inexpensive and allows efficient data collec- tion in the field or laboratory. The design of the tray allows lizards to be positioned for a number of purposes including taking mor- phological and color measurements, photographs, or tissue samples.

Acknowledgments.—We thank J. Van Buskirk for his advice and assis- tance, J. Sinclair, the Live Exhibits staff at Museum Victoria, and two anonymous reviewers. Permits were provided by the Department of Sustainability and Environment Victoria and the Department for Envi- ronment and Heritage South Australia (Permit Nos. 10002952 and E24870, respectively). Funding was provided by an Australian Research Council Discovery Grant (DPO452082) and an Australian Society of Herpetolo- gists Student Research Grant. This research was approved by The Uni- versity of Melbourne Faculty of Science Animal Experimental Ethics Committee and The South Australian Wildlife Ethics Committee.

LITERATURE CITED

HOEFER, A. M., B. A. GOODMAN, AND S. J. DOWNES. 2003. Two effective Corallus hortulanus (Amazon Tree Boa): Two-year old female, un- and inexpensive methods for restraining small lizards. Herpetol. Rev. known locality. Pen-and-ink illustration by Will Brown 34:223-224. (www.blueridgebiological.com).

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