Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13754–13759, December 1997 Evolution The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole (mammalian phylogeneticsyNotoryctesypseudogene) MARK S. SPRINGER*, ANGELA BURK*, JOHN R. KAVANAGH*, VICTOR G. WADDELL†, AND MICHAEL J. STANHOPE† *Department of Biology, University of California, Riverside, CA 92521; and †Biology and Biochemistry, Queens University, 97 Lisburn Road, Belfast BT9 07BL, UK Edited by Roy J. Britten, California Institute of Technology, Corona Del Mar, CA, and approved September 29, 1997 (received for review June 16, 1997) ABSTRACT The subclass Theria of Mammalia includes (ii) dasyuromorphs (3, 10), and (iii) diprotodontians (11, 12). marsupials (infraclass Metatheria) and placentals (infraclass The latter hypothesis derives from single-copy DNA hybrid- Eutheria). Within each group, interordinal relationships remain ization experiments, which also place bandicoots outside of a unclear. One limitation of many studies is incomplete ordinal clade that includes diprotodontians, Dromiciops, and dasyuro- representation. Here, we analyze DNA sequences for part of exon morphs (11, 12). The association of Dromiciops with diprot- 1 of the interphotoreceptor retinoid binding protein gene, in- odontians is inconsistent with the conventional view that cluding 10 that are newly reported, for representatives of all diprotodontians and bandicoots are sister taxa based on the therian orders. Among placentals, the most robust clades are occurrence of syndactyly in these taxa (13, 14). Syndactyly is a Cetartiodactyla, Paenungulata, and an expanded African clade condition in which the second and third digits of the hind foot that includes paenungulates, tubulidentates, and macroscelide- are variably reduced and always are joined together by a ans. Anagalida, Archonta, Altungulata, Hyracoidea 1 Perisso- common integument (13, 14). One implication of the single- dactyla, Ungulata, and the ‘‘flying primate’’ hypothesis are copy DNA results is that syndactyly may not be homologous in rejected by statistical tests. Among marsupials, the most robust diprotodontians and bandicoots and that it evolved indepen- clade includes all orders except Didelphimorphia. The phyloge- dently in these two groups or else was lost in other taxa (e.g., netic placement of the monito del monte and the marsupial mole Dromiciops and dasyuromorphs) after evolving in a more basal remains unclear. However, the marsupial mole sequence con- common ancestor (12, 15). tains three frameshift indels and numerous stop codons in all Another enigmatic taxon is the marsupial mole, Notoryctes three reading frames. Given that the interphotoreceptor retinoid typhlops, which is the only living representative of the order binding protein gene is a single-copy gene that functions in the Notoryctemorphia. Notoryctes possesses numerous autapo- visual cycle and that the marsupial mole is blind with degenerate morphic features related to its fossorial lifestyle and has been eyes, this finding suggests that phenotypic degeneration of the hypothesized to be a relative of diprotodontians (2, 16, 17), eyes is accompanied by parallel changes at the molecular level as dasyurids (18, 19), and bandicoots (3). a result of relaxed selective constraints. Relationships among placental orders are equally contro- versial. At the base of the placental radiation, several studies Marsupials (infraclass Metatheria) and placentals (infraclass suggest that xenarthrans or xenarthrans plus pholidotans are Eutheria) together compose the subclass Theria of Mammalia. the sister group to other placental mammals, collectively Among extant taxa, there are five to seven marsupial orders termed the ‘‘Epitheria’’ (20–26). (1–3) and 18 placental orders (4) (Table 1). Within each Among the epitherian superorders, Archonta, Glires, Pae- subclass, it has proven difficult to resolve higher level rela- nungulata, and Cetartiodactyla have received considerable tionships. Nevertheless, numerous phylogenies have been hy- attention. Archonta (27) includes the orders Chiroptera, Pri- pothesized and serve as a framework for examining questions mates, Scandentia, and Dermoptera. Only a few anatomical in higher level mammal phylogeny. features (e.g., pendulous penis, tarsal specializations) support Among living marsupials, there is uncertainty centering on the the monophyly of this superorder (21, 28). Within this group, earliest cladogenic events. Most workers (1, 2, 5) advocate a Novacek (29) and Simmons (30) suggest an association of fundamental division between American marsupials, excepting chiropterans and dermopterans (5Volitantia) based on mor- the South American microbiothere Dromiciops gliroides (monito phological data, and Pettigrew (31) has proposed a ‘‘flying del monte) and Australasian marsupials plus Dromiciops. The primate’’ hypothesis in which Old World fruitbats (suborder American taxa (Ameridelphia) are united by the presumed Megachiroptera) are more closely related to dermopterans shared derived character of epididymal sperm-pairing (6). Aus- and primates than to microchiropterans. Molecular data pro- tralasian taxa and Dromiciops (Australidelphia), in turn, exhibit a vide some support for the association of Primates, Scandentia, derived morphology of the tarsus that facilitates improved grasp- and Dermoptera together but argue against the inclusion of ing (3, 7). Other hypotheses reject this fundamental split between chiropterans with this group (32–35). ameridelphians and australidelphians and postulate Dromiciops The superorder Glires, which encompasses rodents and (8), caenolestids (3), and didelphids (9), respectively, as the sister lagomorphs, is supported by craniodental and fetal membrane taxon to other living marsupials. characters (22, 36). Morphological data also suggest an asso- Relationships among Australidelphia, assuming monophyly ciation of macroscelideans (elephant shrews) with Glires in the of this group, also are unclear. Dromiciops has been hypoth- superorder Anagalida (37). Glires has been challenged based esized as a sister taxon to (i) all other australidelphians (2, 7), on an analysis of 91 orthologous protein sequences (38). Furthermore, Anagalida is contradicted by both nuclear and The publication costs of this article were defrayed in part by page charge mitochondrial gene studies (35, 39–40). Even the seemingly payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. This paper was submitted directly (Track II) to the Proceedings office. © 1997 by The National Academy of Sciences 0027-8424y97y9413754-6$2.00y0 Abbreviations: IRBP, interphotoreceptor retinoid binding protein; PNAS is available online at http:yywww.pnas.org. ML, maximum likelihood. 13754 Downloaded by guest on October 2, 2021 Evolution: Springer et al. Proc. Natl. Acad. Sci. USA 94 (1997) 13755 Table 1. Classification of therian mammals taining paenungulates, macroscelideans, and tubulidentates. Subclass Theria However, it is questionable whether or not xenarthrans are an Infraclass Metatheria (marsupials) outgroup to other placental mammals (26, 58, 59). Here, we Order Dasyuromorphia (Phascogale 5 phascogale) report 10 IRBP sequences and expand the data set of Stanhope Order Didelphimorphia (Didelphis 5 opossum) et al. (35). The sequences include a hedgehog, a manatee, a Order Diprotodontia (Pseudochirops 5 ringtail possum; pangolin, and seven marsupials. The present collection of Vombatus 5 wombat) sequences is a data set for a nuclear gene that includes Order Microbiotheria (Dromiciops 5 monito del monte) representatives of every placental and marsupial order. This Order Notoryctemorphia (Notoryctes 5 marsupial mole) allows us to examine higher level questions in mammalian Order Paucituberculata (Caenolestes 5 shrew opossum) evolution in the context of a broad phylogenetic framework in Order Peramelina (Echymipera 5 bandicoot) which numerous marsupials and placentals serve as reciprocal Infraclass Eutheria (placentals) outgroups to each other. The inclusion of multiple represen- Order Artiodactyla (Bos 5 cow; Sus 5 pig) tatives from each mammalian infraclass also allows for subdi- Order Carnivora (Felis 5 cat) vision of long branches to minimize the effects of homoplasy. Order Cetacea (Balaenoptera 5 whale; Steno 5 rough-toothed In addition to the phylogenetic implications of the IRBP data, dolphin) we report on the evolution of the IRBP gene in the marsupial Order Chiroptera (Cynopterus 5 dog-faced bat; Megaderma 5 mole, which is blind and has eyes that lack an iris and a lens. false vampire bat; Pteropus 5 flying fox; Tonatia 5 Specifically, indels and stop codons suggest that the Notoryctes round-eared bat) IRBP gene is no longer evolving under purifying selection. Order Dermoptera (Cynocephalus 5 flying lemur) Order Hyracoidea (Procavia 5 hyrax) METHODS Order Insectivora (Erinaceus 5 hedgehogs; Sorex 5 shrew) DNA was extracted from seven marsupials (Caenolestes fuligi- Order Lagomorpha (Oryctolagus 5 rabbit) nosus, Dromiciops gliroides, Echymipera kalubu, Phascogale Order Macroscelidea (Elephantulus 5 elephant shrew) tapoatafa, Notoryctes typhlops, Vombatus ursinus, and Order Perissodactyla (Equus 5 horse) Pseudochirops cupreus) and three placentals (Manis sp., Eri- Order Pholidota (Manis 5 pangolin) naceus europaeus, and Trichechus manatus) as described else- Order