THE RAFFLES BULLETIN OF ZOOLOGY 2012 THE RAFFLES BULLETIN OF ZOOLOGY 2012 60(2): 467–476 Date of Publication: 31 Aug.2012 © National University of Singapore PELVIC-FIN BROODING IN A NEW SPECIES OF RIVERINE RICEFISH (ATHERINOMORPHA: BELONIFORMES: ADRIANICHTHYIDAE) FROM TANA TORAJA, CENTRAL SULAWESI, INDONESIA Fabian Herder Zoologisches Forschungsmuseum Alexander Koenig, Sektion Ichthyologie, Adenauerallee 160, D-53113 Bonn, Germany Email: [email protected] Renny Kurnia Hadiaty Ichthyology Laboratory, Division of Zoology, Research Center for Biology, Indonesian Institute of Sciences (LIPI) JL. Raya Bogor Km 46, Cibinong 16911, Indonesia Email: [email protected] Arne W. Nolte Max-Planck-Institut fuer Evolutionsbiologie, Abteilung Evolutionsgenetik, August-Thienemannstrasse 2, 24306 Ploen Email: [email protected] ABSTRACT. — A new species of ricefi sh is described from a hill stream in Tana Toraja, Sulawesi. Oryzias eversi, new species, is distinguished from all other adrianichthyids in Sulawesi by having a low number of fi n rays in anal (17–18 (19)) and dorsal (10–12) fi ns, only 33–36 scales in lateral midline, ½14 transverse scale rows at dorsal fi n origin, 30–32 (33) vertebrae, small eyes (28.2–35.5% of head length), a conspicuous blackish male courtship colouration, and pelvic brooding behaviour similar to lacustrine ricefi shes. Female Oryzias eversi carry the eggs until the embryos hatch, and show a conspicuous abdominal concavity and extended pelvic fi ns, accommodating and holding the clutch of eggs. The eggs are connected to the female for the whole time of development by attaching fi laments that protrude from the female’s urogenital pore. A mitochondrial haplotype phylogeny suggests that the new species is closely related to another “pelvic brooder”, the lake-dwelling O. sarasinorum from Lake Lindu in Central Sulawesi. However, the haplotype group of O. eversi and O. sarasinorum is nested within a clade of egg-depositing Oryzias from central, southwest, and southeast Sulawesi, whereas another pelvic brooder, Adrianichthys oophorus from Lake Poso, forms a distinct, second lineage of Sulawesi’s ricefi shes. Accordingly, the pelvic brooding strategy has probably evolved more than once and may be realised in river habitats, which represents a new evolutionary trajectory in the radiation of ricefi shes in Sulawesi. The present discovery adds another new ricefi sh species to Sulawesi’s still only partially known ichthyofauna, and highlights the island’s role as hotspot of adrianichthyid diversity. KEY WORDS. — Oryzias, taxonomy, freshwater fi sh, endemism, reproduction, sexual dimorphism INTRODUCTION attaching fi laments (Iwamatsu et al., 2007, 2008; Parenti, 2008). Carrying time varies within and among species, and Ricefi shes (Adrianichthyidae) are small atherinomorph fi shes does not exceed a few hours in most species of the genus with a remarkably diversity of reproductive modes, including Oryzias (Iwamatsu et al., 2007). However, data available ovoviviparity, facultative embryo retention, and maternal care on the reproductive biology of ricefi shes are surprisingly (Parenti, 2005, 2008; Kinoshita et al., 2009). The Indonesian restricted and partly in confl ict, ranging from well-known island Sulawesi is the major hotspot of ricefish species (e.g., for the Medaka, Oryzias latipes, and a few other diversity, harbouring roughly half of the adrianichthyid species that have been investigated so far; Iwamatsu et al., species currently recognised. Most of the island’s endemic 2007, 2008), to poorly known in other species of the family species are restricted to single ancient lakes, i.e., Lake (Kottelat, 1990a, 1990b; Parenti, 2005, 2008; Kinoshita et Poso, Lake Lindu, or the lakes of the Malili Lakes system al. 2009). (Kottelat, 1990a, 1990b; Parenti, 2008; Herder & Chapuis, 2010). In most of the ricefi sh species, females carry small In some species from Sulawesi’s Lakes Poso and Lindu, the bundles of fertilised eggs attached to the maternal fi sh by eggs are reportedly not deposited at all on submerged plants or 467 Herder et al: A new pelvic brooding ricefi sh from Sulawesi other material, but kept by the female until hatching (Kottelat, Germany, and the National Museum of Natural History, 1990a). At least two of these species, Adrianichthys oophorus Smithsonian Institution, Washington, DC (USNM), United from Lake Poso and Oryzias sarasinorum from Lake Lindu, States of America. Morphometric measurements and meristics show clear adaptations for “pelvic brooding” (i.e., females follow Kottelat (1990b) and Parenti & Soeroto (2004). All have extended pelvic fi ns, covering the clutch of eggs kept in a measurements are taken from point to point, recorded to more or less well developed abdominal concavity, attached to the nearest 0.1 mm with a digital caliper. Abbreviations the female by attaching fi laments; Kottelat, 1990a; Iwamatsu used are SL – standard length, HL – head length. Counts of et al., 2007, 2008; Parenti, 2008). As pelvic brooders do not the unpaired fi ns and vertebrae (total = precaudal + caudal) depend on substrate for completing their reproductive cycle, were made from radiographs, using a digital X-ray device this strategy has been considered as a potential adaptation to (Faxitron LX-60). Dorsal- and anal fi n ray counts are reported a predominantly pelagic life style in lake habitats (Kottelat, in total, including the single fi rst unbranched and a number 1990a; Parenti, 2008). In O. sarasinorum, the attaching of branched rays. Principal and procurrent caudal fi n ray fi laments form a “plug” with epithelial cells at the female’s numbers are reported following Rosen & Parenti (1981) and urogenital pore, and there are indications that the developing Parenti (2008). For example, i,4/5,i, stands for one principal embryos affect physiological pathways regulating the unbranched and four branched rays in the dorsal lobe and female’s reproductive physiology (Iwamatsu et al., 2007, fi ve branched and one principal unbranched ray in the ventral 2008); even the possibility of a placental function has been lobe, and 6/7 stands for six procurrent rays in the dorsal and considered (Iwamatsu et al., 2008). seven procurrent rays in the ventral lobe. Scale counts follow Kottelat (1990b); the small scales on caudal fi n base are not Since Parenti’s comprehensive revision of ricefi shes in 2008, included. Scales in transverse row are counted from origin of additional species of Oryzias have been uncovered from dorsal fi n to lateral row and continuing to anal fi n origin; ½ lake and stream habitats in Sulawesi (Herder & Chapuis, refers to the scale in front of the dorsal fi n. Meristic values 2010; Parenti & Hadiaty, 2010; Parenti et al., in prep.). Here in brackets are deviations from the type series present in 4 we describe a new pelvic brooding ricefi sh species from a additional immature specimens examined. forest stream habitat in Tana Toraja (Central Sulawesi, South Phylogenetic analyses. — Complementary to ricefish Sulawesi Province), the fi rst record of this reproductive sequence data available from Takehana et al. (2005), a 16s strategy (or reproductive guild sensu Kottelat, 1990a) in a rDNA fragment was sequenced using the primers L2606 5'- stream habitat. As this discovery raises questions concerning TTGACCGTGCAAAGGTAGCGCAATCACT-3' and H3056 the origin and evolutionary ecology of pelvic brooding, we 5'-CTCCGGTCTGAACTCAGATCACGTAGG-3' from the present a mitochondrial haplotype phylogeny of Sulawesi’s same authors for Oryzias eversi and additional adrianichthyids ricefi shes and discuss the evolutionary origins of pelvic from Sulawesi (Appendix 1). DNA was extracted using brooding. The present results provide further support for DNeasy Blood & Tissue Kit (Quiagen) and PCR amplifi ed the still “under-described” (Roberts, 1998: 213) status of using the Quagen Multiplex PCR Kit following the protocols ricefi shes, also highlighted by the recent discovery of new supplied by the provider. The following PCR program was species from other Asian regions (Magtoon, 2010; Asai et used: 15 min at 95°C, 36 cycles of (30 s at 94°C, 90 s at 68°C, al., 2011), and further emphasize our incomplete knowledge 90 s at 72°C) and a fi nal extension of 10 min at 72°C. Products of the freshwater ichthyofauna of Sulawesi. for sequencing were prepared for sequencing using an exo- sap protocol and sanger-sequenced in both directions on an ABI 3130xl Genetic Analyser. Sequences of representative MATERIAL AND METHODS adrianichthyid taxa from outside of Sulawesi and additional sequence data available for Sulawesi ricefi shes were retrieved Analyses of morphology and reproductive behaviour. — from genbank (http://www.ncbi.nlm.nih.gov/genbank/; see Specimens of the new Oryzias were obtained from a tributary Appendix 1). All sequences were aligned using BioEdit (Hall, of Salo Sadang (3°02.126S, 119°53.232E) about 8 km south 1999) and electropherograms and alignments were checked of Rantepao, Tana Toraja, Central Sulawesi, Indonesia, in by eye and edited after combining the data with reference 24 Sep.2010. Fish were caught by snorkeling using hand sequences from GenBank. The fi nal data was trimmed to nets. Single individuals of both sexes were photographed 477 bp. We used MrModeltest (Nylander, 2004) to infer the immediately after capture to document life colouration; a stock most likely model of sequence evolution and used MrBayes of living individuals was kept alive in aquaria to investigate (Ronquist et al., in press) to infer a bayesian phylogeny and reproductive