Neuroendocrine peptides (insulin, pancreatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide ) from the desert tortoise, Gopherus agassizii Yuqi Wang a , Valentine A. Lance b , Per F. Nielsen c , J. Michael Conlon a, * a Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA b Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA 92112, USA c Novo Nordisk A/S, Health Care Discovery, 2880 Bagsvaerd, Denmark Received 4 December 1998; accepted 1 February 1999 Abstract The traditional view that Testudines (tortoises and turtles) should be regarded as the surviving clade of the anapsid reptiles rather than classified with the diapsid reptiles (snakes, lizards, and crocodiles) has recently been challenged. Neuropeptide Y, neuropeptide , and somatostatin-14 were isolated from an extract of the brain, substance P and galanin from an extract of the intestine, and insulin and pancreatic polypeptide from an extract of the pancreas of the desert tortoise, Gopherus agassizii. Despite that crocodilians did not appear until the late Triassic, the amino acid sequences of the tortoise peptides resemble those of the American alligator quite closely. The primary structures of neuropeptide Y, somatostatin-14, and neuropeptide  are the same in tortoise and alligator. The primary structures of substance P, insulin, galanin, and pancreatic polypeptide in the two species differ by 1, 3, 5, and 8 amino acid residues, respectively. Although fewer neurohormonal peptides from squamates (lizards and snakes) have been characterized, the primary structures of neuropeptide , insulin, and pancreatic polypeptide from the Burmese python and the desert tortoise differ by 3, 8, and 18 residues, respectively. The data suggest, therefore, a closer phylogenetic relationship between Testudines and Crocodilians than that derived from ‘classical’ analyses based on morphological criteria and the fossil record. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Gastrointestinal neuropeptides; Insulin; Pancreatic polypeptide; Reptilian phylogeny 1. Introduction Immunohistochemical studies have shown that the pancreata and gastrointestinal tracts of all classes of reptile yet studied contain the complex distribution of most, if not all, of the regulatory peptides found in mammals (for review, see Conlon et al. [4,7]). For ex- ample, in the intestine of the turtle, Chrysemys picta, mucosal endocrine cells of the open-type reacted with antisera to gastrin, somatostatin, peptide tyrosine– tyrosine (PYY), secretin, neurotensin, motilin, gastric inhibitory polypeptide, and glucagon. Pancreatic endo- crine cells were stained immunocytochemically for insulin, glucagon, somatostatin, and pancreatic polypep- tide (PP) [12]. However, our knowledge of the primary structures of neuroendocrine peptides from reptilian species is fragmentary and studies have focused on crocodilians and snakes. Two forms of gonadotropin- releasing hormone [19] and neuropeptide Y (NPY) [29] have been purified from alligator brain and tachykinin- related peptides corresponding to mammalian substance P, neurokinin A (NKA), and neuropeptide  have been isolated from the brain of the alligator [31] and intes- tine of the python [4]. Gastrin-releasing peptide [29], vasoactive intestinal polypeptide [29], somatostatin-14 [29], and galanin [29] have been purified from an extract of alligator stomach and neurotensin from the intestine of both the alligator [26] and python [4]. The primary structure of insulin is known for the alligator [17] and three species of snake [7,15,34]. Our knowledge of the structures of neurohormonal peptides from Testudines is confined to the structures of the pancreatic hormones * Corresponding author. Tel.: +1-402-280-1733; fax: +1-402-280- 2690. E-mail address: jmconlon@creighton.edu (J.M. Conlon) Peptides 20 (1999) 713–722 0196-9781/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0196-9781(99)00053-4