Skip to main content Skip to main navigation menu Skip to site footer
Type: Article
Published: 2016-06-24
Page range: 515–536
Abstract views: 97
PDF downloaded: 1

Larval morphology and complex vocal repertoire of Rhacophorus helenae (Anura: Rhacophoridae), a rare flying frog from Vietnam

Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP–1, Moscow 119234, Russia Joint Russian-Vietnamese Tropical Research and Technological Center under the A.N. Severtsov Institute of Ecology and Evolution RAS, South Branch, 3, Street 3/2, 10 District, Ho Chi Minh City, Vietnam
Joint Russian-Vietnamese Tropical Research and Technological Center under the A.N. Severtsov Institute of Ecology and Evolution RAS, South Branch, 3, Street 3/2, 10 District, Ho Chi Minh City, Vietnam Zoological Museum of the Lomonosov Moscow State University, Bolshaya Nikitskaya st. 6, Moscow 125009, Russia
Department of Vertebrate Zoology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory, GSP–1, Moscow 119234, Russia Joint Russian-Vietnamese Tropical Research and Technological Center under the A.N. Severtsov Institute of Ecology and Evolution RAS, South Branch, 3, Street 3/2, 10 District, Ho Chi Minh City, Vietnam
Amphibia amphibians advertisement call DNA barcoding mouthparts Rhacophorus reinwardtii species complex Southeast Asia tadpole

Abstract

We present new data on the distribution, reproduction, larval morphology and vocalization of Rhacophorus helenae (Rhacophoridae), a narrowly distributed frog from southern Vietnam. Two new populations of R. helenae were discovered during field surveys in the lowland monsoon forests in Dong Nai and Ba Ria–Vung Tau provinces in 2010–2013. Spawning was observed in May 2013. Egg clutches containing small (2.3±0.1 mm) unpigmented eggs were embedded in a foam nest and suspended high on trees above temporary ponds. The tadpoles of R. helenae have a morphology typical of pond-dwelling Rhacophorus larvae with a moderate tail length and a labial tooth row formula of 5(2–5)/3. Postmetamorphic juveniles differed from adult frogs in the features of their coloration and less developed webbing. The complex vocal repertoire of R. helenae included five types of tonal, wideband and pulsed calls and several transitional signal types differentiated by frequency and amplitude parameters. Calls were uttered as singular signals (pulsed calls) or within non-stereotyped series of variable duration (other call types). The complex structure of the advertisement call markedly distinguishes R. helenae from other members of the Rhacophorus reinwardtii species complex.

 

References

  1. Altig, R. (1970) A key to the tadpoles of Continental United States and Canada. Herpetologica, 26, 180–207.

    Altig, R. & McDiarmid, R.W. (2007) Morphological diversity and evolution of egg and clutch structure in amphibians. Herpetological Monographs, 21, 1–32.
    http://dx.doi.org/10.1655/06-005.1

    Bee, M.A., Suyesh, R. & Biju, S.D. (2013a) Vocal behavior of the Ponmudi bush frog (Raorchestes graminirupes): repertoire and individual variation. Herpetologica, 69, 22–35.
    http://dx.doi.org/10.1655/HERPETOLOGICA-D-11-00042

    Bee, M.A., Suyesh, R. & Biju, S.D. (2013b) The vocal repertoire of Pseudophilautus kani, a shrub frog (Anura: Rhacophoridae) from the Western Ghats of India. Bioacoustics, 22, 67–85.
    http://dx.doi.org/10.1080/09524622.2012.712750

    Berry, P.Y. (1972) Undescribed and little known tadpoles from West Malaysia. Herpetologica, 28, 338–346.

    Chan, K.O. & Grismer, L.L. (2010) Re-assessment of the Reinwardt’s Gliding Frog, Rhacophorus reinwardtii (Schlegel 1840) (Anura: Rhacophoridae) in southern Thailand and peninsular Malaysia and its re-description as a new species. Zootaxa, 2505, 40–50.

    Chou, W.-H. & Lin, J.-Y. (1997) Tadpoles of Taiwan. Special Publication National Museum of Natural Science, 7, 1–98.

    Christensen-Dalsgaard, J., Ludwig, T.A. & Narins, P. (2002) Call diversity in an Old World treefrog: Level dependence and latency of acoustic responses. Bioacoustics, 13, 21–35.
    http://dx.doi.org/10.1080/09524622.2002.9753484

    De Koninck, R. (1999) Deforestation in Viet Nam. International Research Centre, Ottawa, 110 pp.

    Dring, J.C.M. (1979) Amphibians and reptiles from northern Terengganu, Malaysia, with descriptions of two new geckos: Cnemaspis and Cyrtodactylus. Bulletin of the British Museum Natural History (Zoology), 34, 181–241.

    Drummond, A.J. & Rambaut, A. (2007) Beast: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7, 214.
    http://dx.doi.org/10.1186/1471-2148-7-214

    Duellman, W. & Trueb, L. (1994) Biology of Amphibians. Johns Hopkins University Press, Baltimore, MD, 670 pp.

    Felsenstein, J. (1985a) Evolutionary trees from DNA sequences: A maximum likelihood approach. Journal of Molecular Evolution, 17, 368–376.
    http://dx.doi.org/10.1007/BF01734359

    Felsenstein, J. (1985b) Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791.
    http://dx.doi.org/10.2307/2408678

    Gerhardt, H.C. & Huber, F. (2002) Acoustic Communication in Insects and Anurans: Common Problems and Diverse Solutions. University of Chicago Press, Chicago, 531 pp.

    Gosner, K.L. (1960) A simplified table for staging anuran embryos and larvae. Herpetologica, 16, 183–190.

    Grosjean, S. (2001) The tadpole of Leptobrachium (Vibrissaphora) echinatum (Amphibia, Anura, Megophryidae). Zoosystema, 23, 143–156.

    Grosjean, S., Delorme, M., Dubois, A. & Ohler, A. (2008) Evolution of reproduction in the Rhacophoridae (Amphibia, Anura). Journal of Zoological Systematics and Evolutionary Research, 46, 169–176.
    http://dx.doi.org/10.1111/j.1439-0469.2007.00451.x

    Grosjean, S., Glos, J., Teschke, M., Glaw, F. & Vences, M. (2007) Comparative larval morphology of Madagascan toadlets of the genus Scaphiophryne: phylogenetic and taxonomic inferences, Zoological Journal of the Linnean Society, 151, 555–576.
    http://dx.doi.org/10.1111/j.1096-3642.2007.00329.x

    Grosjean, S., Ohler, A., Chuaynkern, Y., Cruaud, C. & Hassanin, A. (2015) Improving biodiversity assessment of anuran amphibians using DNA barcoding of tadpoles. Case studies from Southeast Asia. Comptes Rendus Biologies, 338, 351–361.
    http://dx.doi.org/10.1016/j.crvi.2015.06.001

    Grosjean, S., Randrianiaina, R.-D., Strauss, A. & Vences, M. (2011a) Sand-eating tadpoles in Madagascar: morphology and ecology of the unique larvae of the treefrog Boophis picturatus. Salamandra, 47, 63–76.

    Grosjean, S., Strauss, A., Glos, J., Randrianiaina, R.D., Ohler, A. & Vences, M. (2011b) Morphological and ecological uniformity in the funnel-mouthed tadpoles of Malagasy litter frogs, subgenus Chonomantis. Zoological Journal of the Linnean Society, 162, 149–183.
    http://dx.doi.org/10.1111/j.1096-3642.2010.00667.x

    Gillespie, G.R., Anstis, M., Howard, S.D. & Lockie, D. (2007) Description of the tadpole of the Rhacophorid frog Rhacophorus georgii Roux (Rhacophoridae) from Sulawesi, Indonesia. Journal of Herpetology, 41, 150–153.
    http://dx.doi.org/10.1670/0022-1511(2007)41[150:DOTTOT]2.0.CO;2

    Haas, A., Hertwig, S.T., Krings, W., Braskamp, E., Dehling, J.M., Min, P.Y., Jankowski, A., Schweizer, M. & Das, I. (2012) Description of three Rhacophorus tadpoles (Lissamphibia: Anura: Rhacophoridae) from Sarawak (Borneo). Zootaxa, 3328, 1–19.

    Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.

    Hedges, S.B. (1994) Molecular evidence for the origin of birds. Proceedings of the National Academy of Sciences of the United States of America, 91, 2621–2624.
    http://dx.doi.org/10.1073/pnas.91.7.2621

    Hendrix, R., Grosjean, S., Le, Q.K., Vences, M., Vu, T.N. & Ziegler, T. (2007) Molecular identification and description of the tadpole of the Annam Flying frog, Rhacophorus annamensis SMITH, 1924 (Anura : Rhacophoridae). Salamandra, 43, 11–19.

    Hillis, D.M, Moritz, C. & Mable, B.K (1996) Molecular Systematics. 2nd edition. Sinauer Associates, Inc, Sunderland, Massachusetts, xvi + 655 pp.

    Huelsenbeck, J.P. & Hillis, D.M. (1993) Success of phylogenetic methods in the four-taxon case. Systematic Biology, 42, 247–264.
    http://dx.doi.org/10.2307/2992463

    Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics, 17, 754–755.
    http://dx.doi.org/10.1093/bioinformatics/17.8.754

    Inger, R.F. (1985) Tadpoles of the forested regions of Borneo. Fieldiana: Zoology, New Series, 26, i–v+1–89.

    Inger, R.F. & Stuebing, R.B. (2005) A Field Guide to the Frogs of Borneo. 2nd Edition. Natural History Publications (Borneo), Kota Kinabalu, Sabah, 201 pp.

    IUCN SSC Amphibian Specialist Group (2014) Rhacophorus helenae. The IUCN Red List of Threatened Species 2014: e.T47144519A47144530.
    http://dx.doi.org/10.2305/IUCN.UK.2014-1.RLTS.T47144519A47144530.en

    Jamieson, B.G.M. (2003) Reproductive Biology and Phylogeny of Anura. Science Publishers, Inc., Enfield, NH, 452 pp.

    Jiang, K., Yan, F., Suwannapoom, C., Chomdej, S. & Che, J. (2013) A new species of the genus Leptolalax (Anura: Megophryidae) from northern Thailand. Asian Herpetological Research, 4, 100–108.
    http://dx.doi.org/10.3724/SP.J.1245.2013.00100

    Jobb, G., von Haeseler, A. & Strimmer, K. (2004) TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics. BMC Evolutionary Biology, 4, 18.
    http://dx.doi.org/10.1186/1471-2148-4-18

    Kasuya, E., Kobayashi, T., Ootaki, M., Oota, N. & Takada, A. (1997) Female preference for temporal features of vocalization in the Japanese treefrog, Rhacophorus arboreus. Journal of Ethology, 15,103–108.
    http://dx.doi.org/10.1007/BF02769395

    Kasuya, E., Kumaki, T. & Saito, T. (1992) Vocal repertoire of the Japanese treefrog, Rhacophorus arboreus. Zoological Science, 9, 469–473.

    Kime, N.M., Turner, W.R. & Ryan, M.J. (2000) The transmission of advertisement calls in Central American frogs. Behavioral Ecology, 11, 71–83.
    http://dx.doi.org/10.1093/beheco/11.1.71

    Laurance, W.F. (2007) Forest destruction in tropical Asia. Current Science, 93, 1544–1550.

    Liem, S.S. (1970) The morphology, systematics, and evolution of the Old World treefrogs (Rhacophoridae and Hyperoliidae). Fieldiana Zoology, 57, 1–145.
    http://dx.doi.org/10.5962/bhl.title.2939

    Littlejohn, M.J. (1977) Long-range acoustic communication in anurans: an integrated and evolutionary approach. In: Taylor, D.H. & S.I. Guttman, S.I. (Eds.), The reproductive biology of amphibians. Plenum, New York, pp. 263–94.
    http://dx.doi.org/10.1007/978-1-4757-6781-0_8

    Manthey, U. & Grossmann, W. (1997) Amphibien und Reptilien Südostasiens. Natur und Tier Verlag, Münster, 512 pp.

    Matsui, M., Chen, S.-L. & Lue, K.-Y. (1997) Advertisement call characteristics of a Taiwanese green treefrog Rhacophorus prasinatus. Bonner Zoologische Beiträge, 47, 165–174.

    Matsui, M., Shimada, T. & Sudin, A. (2013) A new gliding frog of the genus Rhacophorus from Borneo. Current Herpetology, Kyoto, 32, 112–124.
    http://dx.doi.org/10.5358/hsj.32.112

    Matsui, M. & Wu, G.-F. (1994) Acoustic characteristics of treefrogs from Sichuan, China, with comments on systematic relationship of Polypedates and Rhacophorus (Anura, Rhacophoridae). Zoological Science, 11, 485–490.

    McDiarmid, R.W. & Altig, R. (1999) Tadpoles: The Biology of Anuran Larvae. University of Chicago Press, Chicago, 458 pp.

    Meijer, W. (1973) Devastation and Regeneration of Lowland Dipterocarp Forests in Southeast Asia. BioScience, 23, 528–533.
    http://dx.doi.org/10.2307/1296481

    Meyfroidt, P. & Lambin, E.F. (2008) Forest transition in Vietnam and its environmental impacts. Global Change Biology, 14, 1319–1336.
    http://dx.doi.org/10.1111/j.1365-2486.2008.01575.x

    Murphy, R.W., Crawford, A.J., Bauer, A.M., Che, J., Donnellan, S.C., Fritz, U., Haddad, C.F.B., Nagy, Z.T., Poyarkov, N.A., Jr., Vences, M., Wang, W.-Z. & Zhang, Y.-P. (2013). Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles. Molecular Ecology Resources, 13, 161–167.
    http://dx.doi.org/10.1111/1755-0998.12050

    Nguyen, N.H. & Hoang, M.D. (2013) The species composition of Amphibian and Reptile in Binh Chau–Phuoc Buu Nature Reserve, Ba Ria Vung Tau Province. In: Proceedings of the Fifth National Conference on Ecology and Biological Resources. Agriculture Publishing House, Hanoi, pp. 504–513.

    Ohler, A. & Delorme, M. (2006) Well known does not mean well studied: Morphological and molecular support for existence of sibling species in the Javanese gliding frog Rhacophorus reinwardtii (Amphibia, Anura). Comptes Rendus Biologies, 329, 86–97.
    http://dx.doi.org/10.1016/j.crvi.2005.11.001

    Ohler, A., Wollenberg, K.C., Grosjean, S., Hendrix, R., Vences, M., Ziegler, T. & Dubois, A. (2011) Sorting out Lalos: description of new species and additional taxonomic data on megophryid frogs from northern Indochina (genus Leptolalax, Megophryidae, Anura). Zootaxa, 3147, 1–83.

    Pope, C.H. (1931) Notes on the amphibians from Fukien, Hainan, and other parts of China. The Bulletin of the American Museum of Natural History, 61, 397–611.

    Posada, D. & Crandall, K.A. (1998) Modeltest: testing the model of DNA substitution. Bioinformatics, 14, 817–818.
    http://dx.doi.org/10.1093/bioinformatics/14.9.817

    Poyarkov, N.A. Jr., Orlov, N.L., Moiseeva, A.V., Pawangkhanant, P., Ruangsuwan, T., Vassilieva, A.B., Galoyan, E.A., Nguyen, T.T. & Gogoleva, S.S. (2015a) Sorting out moss frogs: mtDNA data on taxonomic diversity and phylogenetic relationships of the Indochinese species of the genus Theloderma (Anura, Rhacophoridae). Russian Journal of Herpetology, 22, 241–280.

    Poyarkov, N.A. Jr., Rowley, J.J.L., Gogoleva, S.S., Vassilieva, A.B., Galoyan, E.A. & Orlov, N.L. (2015b) A new species of Leptolalax (Anura: Megophryidae) from the western Langbian Plateau, southern Vietnam. Zootaxa, 3931 (2), 221–252.
    http://dx.doi.org/10.11646/zootaxa.3931.2.3

    Preininger, D, Böeckle, M. & Hödl, W. (2007) Comparison between anuran acoustic communities of two habitat types in the Danum Valley Conservation Area, Sabah, Malaysia. Salamandra, 43,129–138.

    Rand, A.S. & Ryan, M.J. (1981) The Adaptive Significance of a Complex Vocal Repertoire in a Neotropical Frog. Zeitschrift für Tierpsychologie, 57, 209–214.
    http://dx.doi.org/10.1111/j.1439-0310.1981.tb01923.x

    Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574.
    http://dx.doi.org/10.1093/bioinformatics/btg180

    Rowley, J.J.L., Dau, Q.V., Hoang, H.D., Nguyen, T.T., Le, D.T.T. & Altig, R. (2015) The breeding biologies of three species of treefrogs with hyperextended vocal repertoires (Gracixalus; Anura: Rhacophoridae). Amphibia-Reptilia, 36, 277–285.
    http://dx.doi.org/10.1163/15685381-00003007

    Rowley, J.J.L., Dau, Q.V., Nguyen, T.T., Cao, T.T. & Nguyen, S.V. (2011a) A new species of Gracixalus (Anura: Rhacophoridae) with a hyperextended vocal repertoire from Vietnam. Zootaxa, 3125, 22–38.

    Rowley, J.J.L., Le, D.T.T., Hoang, H.D., Dau, V.Q. & Cao, T.T. (2011b) Two new species of Theloderma (Anura: Rhacophoridae) from Vietnam. Zootaxa, 3098, 1–20.

    Rowley, J.J.L., Tran, D.T.A., Hoang, H.D. & Le, D.T.T. (2012a) A new large species of large Flying Frog (Rhacophoridae: Rhacophorus) from lowland forests of southern Vietnam. Journal of Herpetology, 46, 480–487.
    http://dx.doi.org/10.1670/11-261

    Rowley, J.L., Dao, T.T.A., Duong, L.T.T., Huy, H.D. & Altig, R. (2012b) The strangest tadpole: the oophagous, tree-hole dwelling tadpole of Rhacophorus vampyrus (Anura: Rhacophoridae) from Vietnam. Journal of Natural History, 46, 2969–2978.
    http://dx.doi.org/10.1080/00222933.2012.732622

    Ryan, M.J. (2001) Anuran communication (Ed.) Smithsonian Institution Press, Washington, DC, 252 pp.

    Sheridan, J.A., Bickford, D. & Su, K.F.-Y. (2010) An examination of call and genetic variation in three wide-ranging Southeast Asian anuran species. Raffles Bulletin of Zoology, 58, 369–379.

    Smith, A.M., Poyarkov, N.A. Jr. & Hebert, P.D.N. (2008) CO1 DNA barcoding amphibians: take the chance, meet the challenge. Molecular Ecology Notes, 8, 235–246.
    http://dx.doi.org/10.1111/j.1471-8286.2007.01964.x

    Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731–2739.
    http://dx.doi.org/10.1093/molbev/msr121

    Thomas, M., Raharivololoniaina, L., Glaw, F., Vences, M. & Vieites, D.R. (2005) Montane tadpoles in Madagascar: molecular identification and description of the larval stages of Mantidactylus elegans, Mantidactylus madecassus, and Boophis laurenti from the Andringitra Massif. Copeia, 2005, 174–183.
    http://dx.doi.org/10.1643/CH-03-293R2

    Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25 (24), 4876–4882.
    http://dx.doi.org/10.1093/nar/25.24.4876

    Trepanier, T.L., Lathrop, A. & Murphy, R.W. (1999) Rhacophorus leucomystax in Vietnam with acoustic analyses of courtship and territorial calls. Asiatic Herpetological Research, 8, 102–106.

    Vassilieva, A.B., Galoyan, E.A., Gogoleva, S.S. & Poyarkov, N.A., Jr. (2014) Two new species of Kalophrynus Tschudi, 1838 (Anura: Microhylidae) from the Annamite mountains in southern Vietnam. Zootaxa, 3796 (3), 401–434.
    http://dx.doi.org/10.11646/zootaxa.3796.3.1

    Vassilieva, A.B., Galoyan, E.A. & Poyarkov, N.A. (2013) Rhacophorus vampyrus (Anura: Rhacophoridae) Reproductive Biology: A New Type of Oophagous Tadpole in Asian Treefrogs. Journal of Herpetology, 47, 607–614.
    http://dx.doi.org/10.1670/12-180

    Vences, M., Thomas, M., Bonett, R.M. & Vieites, D.R. (2005a) Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philosophical Transactions of the Royal Society London, Series B, 360, 1859–1868.
    http://dx.doi.org/10.1098/rstb.2005.1717

    Vences, M., Thomas, M., van der Meijden, A., Chiari Y. & Vieites, D.R. (2005b) Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology, 2, 5.
    http://dx.doi.org/10.1186/1742-9994-2-5

    Vieites, D.R., Wollenberg, K.C., Andreone, F., Kohler, J., Glaw, F. & Vences, M. (2009) Vast underestimation of Madagascar’s biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences of the United States of America, 106, 8267–8272.
    http://dx.doi.org/10.1073/pnas.0810821106

    Wells, K.D. (2007) The Ecology and Behaviour of Amphibians. Chicago University Press, 1148 pp.
    http://dx.doi.org/10.7208/chicago/9780226893334.001.0001

    Wells, K.D.(1988) The effect of social interactions on anuran vocal behavior. In: Fritzsch, B., Ryan, M.J., Wilczynski, W., Hetherington, T.E. & Walkowiak, W. (Eds.), The evolution of the amphibian auditory system. Wiley, New York, pp. 433–454.

    Wildenhues, M.J., Bagaturov, M.F., Schmitz, A., Dao, T.T.A., Hendrix, R. & Ziegler, T. (2011) Captive management and reproductive biology of Orlov’s treefrog, Rhacophorus orlovi Ziegler & Köhler, 2001 (Amphibia: Anura: Rhacophoridae), including larval description, colour pattern variation and advertisement call. Der Zoologische Garten, 80, 287–303.
    http://dx.doi.org/10.1016/j.zoolgart.2011.10.002

    Wildenhues, M.J., Gawor, A., Nguyen, T.Q., Nguyen, T.T., Schmitz, A. & Ziegler, T. (2010) First description of larval and juvenile stages of Rhacophorus maximus Günther, 1859 1858 (Anura: Rhacophoridae) from Vietnam. Revue suisse de Zoologie, 117, 679–696.
    http://dx.doi.org/10.5962/bhl.part.118618

    Xia, Y., Gu, H.-F., Peng, R., Chen, Q., Zheng, Y.-C., Murphy, R.W. & Zeng, X.-M. (2012) COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae). Molecular Ecology Resources, 12, 48–56.
    http://dx.doi.org/10.1111/j.1755-0998.2011.03055.x

    Zheng, Y., Li, S. & Fu, J. (2008) A phylogenetic analysis of the frog genera Vibrissaphora and Leptobrachium, and the correlated evolution of nuptial spine and reversed sexual size dimorphism. Molecular Phylogenetics and Evolution, 46, 695–707.
    http://dx.doi.org/10.1016/j.ympev.2007.09.019

    Ziegler, T. (2002) Die Amphibien und Reptilien eines Tieflandfeuchtwald-Schutzgebietes in Vietnam. Natur und Tier Verlag, Münster, 342 pp.