II ||I] TEXAS TECH UNIVERSITY Natural Science Research Laboratory

Special Publications Museum of Texas Tech University Number 49 1 September 2006

Molecular and Morphological Analyses of the Sportive (Family Megaladapidae: Genus Lepilemur) Reveals 11 Previously Unrecognized Species

Edward E. Louis, Jr., etal. Front cover: Current distribution of the sportive lemurs of based on molecular data. Figure prepared by Kelly Herrington, Shannon Engberg, and Runhua Lei. SPECIAL PUBLICATIONS

Museum of Texas Tech University Number 49

Molecular and Morphological Analyses of the Sportive Lemurs (Family Megaladapidae: Genus Lepilemur) Reveals 11 Previously Unrecognized Species

Edward E. Louis, Jr., Shannon E. Engberg, Runhua Lei’ Huimin Geng, Julie A. Sommer, Richard Randriamampionona, Jean C. Randriamanana, John R. Zaonarivelo, Rambinintsoa Andriantompohavana, Gisele Randria, Prosper, Borome Ramaromilanto, Gilbert Rakotoarisoa, Alejandro Rooney, and Rick A. Brenneman

Henry Doorly Zoo, University of Nebraska Medical Center and School of Biological Sciences, University of Antananarivo, Parc Botanique et Zoologique de Tsimbazaza, and US. Department of Agriculture Layout and Design: Jacqueline Chavez Cover Design: Kelly Herrington, Shannon Engberg, and Runhua Lei

Copyright 2006, Museum of Texas Tech University

All rights reserved. No portion of this book may be reproduced in any form or by any means, including electronic storage and retrieval systems, except by explicit, prior written permission of the publisher.

This book was set in Times New Roman and printed on acid-free paper that meets the guidelines for permanence and durability of the Committee on Production Guidelines for Book Longevity of the Council on Library Resources.

Printed: 1 September 2006

Library of Congress Cataloging-in-Publication Data

Special Publications of the Museum of Texas Tech University, Number 49 Series Editor: Robert J. Baker

Molecular and Morphological Analyses of the Sportive Lemurs (Family Megaladapidae: Genus Lepilemur) Reveals 11 Previously Unrecognized Species

Edward E. Louis, Jr., Shannon E. Engberg, Runhua Lei, Huimin Geng, Julie A. Sommer, Richard Randriamampionona, Jean C. Randriamanana, John R. Zaonarivelo, Rambinintsoa Andriantompohavana, Gisele Randria, Prosper, Borome Ramaromilanto, Gilben Rakotoarisoa, Alejandro Rooney, and Rick A. Brenneman

ISSN 0169-0237 ISBN 1-929330-10-3

Museum of Texas Tech University Lubbock, TX 79409-3191 USA (806)742-2442 Molecular and Morphological Analyses of the Sportive Lemurs (Family Megaladapidae: Genus Lepilemur) Reveals 11 Previously Unrecognized Species

Edward E. Louis, Jr., Shannon E. Engberg, Runhua Lei, Huimn Genu, Julie A. Sommer, Richard Randriamampionona, Jean C. Randriamanana, John R. Zaonarjvelo, Rambinintsoa Andriantompoha van a, Gisele Randrja, Prosper, Borome Ramaromilanto, Gilbert Rakotoarisoa, Alejandro Rooney, and RjckA. Brenneman

Abstract

Approximately 3,800 base pairs of mitochondrial DNA sequence data (control region or D-loop, 12s ribosomal RNA subunit gene, along with a fragment including a partial segment of cytochrome oxidase subunit 111 gene to Leucine-tRNA gene) were used to investigate the phylogenetic relationships among the sportive lemurs (Genus Lepilemur) and to other genera of Madagascar. The results of our analyses support a monotypic relationship of Lepilemur species to other lemur genera, with a sister relationship to the Family . Most importantly, on the basis of both molecular sequence and morphological data, our results show the existence of at least 11 previously unrecognized species of Lepilemur. The phylogenetic analyses presented confirm a westem/eastem split among the Lepilemur species with the exception of the Small-toothed found at Ranomafana and Tolongoina, which is included within the western clade. These results underscore the urgency to initiate further de¬ tailed studies in previously unstudied sites throughout Madagascar in order to better define lemur species.

Key words: conservation, lemur, Lepilemur, phylogeography, systematics

Introduction

Due to its unique species and to The taxonomic revision of species and distribu¬ the continued pressure from human encroachment, tions warrants the need to consistently re-evaluate Madagascar has been placed at the top of conservation the conservation protection status of lemurs as new priority lists, or hotspots (Myers 2000). Distributed information becomes available (Martin 2000). Recent throughout the island, lemurs are particularly suscep¬ molecular genetic and morphological studies within the tible to risks due to their relatively small genera Avahi, Cheirogateus, Lepilemur. and Microce- fragmented geographic ranges (Jemvall and Wright bus have led to a significant increase in the number 1998). All lemurs are protected under the Conven¬ of recognized species (Andriaholinirina et al. 2006; tion of International Trade of Endangered Species Groves 2000; Kappeler et al. 2005; Louis et al. 2006; (CITES) and are designated by the IUCN/SSC Red Rasoloarison et al. 2000; Thalmann and Geissmann List Categories from critically endangered to threatened 2005; Zimmermann et al. 1998). Thus, it is critical depending on the species (IUCN/SSC 1999). Thus, that we accurately define species and subspecies to there is a pressing need to understand the better evaluate conservation risks and make appropri¬ and phylogeny of lemurs so that a scientifically rational ate recommendations for the management of wild approach to their conservation and management can be populations, especially when considering the ranges developed and implemented. of newly recognized species of lemurs. The sportive

1 2 Special Publications, Museum of Texas Tech University lemurs (Genus Lepilemur), a group of strictly nocturnal standard, Thalmann and Geissmann (2005) presented that are superficially indistinguishable with an alternative approach through the use of only morpho¬ varying degrees of brown, red, white, and gray pelage, logic data, detailed photographs, and tissue samples. are a prime example of species that until now have been The endangered status of lemurs, all of which have been largely unrecognized and underrepresented. designated CITES appendix I, along with the digital and molecular technological capabilities of the twenty-first The only member of the monotypic Fam¬ century, can eliminate the immediate requirement of ily Megaladapidae, the Genus Lepilemur is currently whole vouchers from the wild. Nonetheless, whole represented by 11 extant species broadly distributed vouchers can subsequently supplement the process as across Madagascar (Appendix Ib).The taxonomy of the they become available opportunistically (e.g., raptor sportive lemurs has been revised several times (Groves nests or remains of Fossa [Cryptoprocta ferox] preda¬ 2001; Ishak et al. 1992; Jenkins 1987; Mittermeier et tion). Due to the inability to maintain sportive lemurs al. 1994; Mittermeier et al. 2006; Montagnon et al. as long-term live vouchers in captivity, the type series 2001; Petter and Petter-Rousseaux 1960: Petter et al. for these new ly described Lepilemur species are repre¬ 1977; Ravaoarimanana 2001; Ravaoarimanana et al. sented by whole blood samples from free-ranging indi¬ 2004; Rumpler et al. 2001; Rumpler and Albignac viduals along with a database containing all field data, 1975; Schwarz 1931; Tattersall 1982; Thalmann 2000; accessioned sequence data, and photographs. The type Thalmann and Ganzhorn 2003), most recently by An- series tissues and database are curated at The Museum driaholinirina et al. (2006) who described three new of Texas Tech University (TK 125501-125566/TTU species. Here, we present further taxonomic revisions 104434-104499) (Appendix II). by describing 11 new species. We also conducted a phylogenetic analysis of the relationships between The database referenced above, as well as addi¬ these new species and previously recognized ones of tional tables and figures, also are available online at the the genus Lepilemur. website of Omaha’s Henry Doorly Zoo. See Appendix I for a directory of the appropriate website addresses. Although adopting a species description meth¬ odology that required a whole voucher has been the

Methods

Sampling.—All lemurs investigated in this study We present the weight, head crown, body length, and were free-ranging and were immobilized with a CO, tail length in this publication following the guidelines of projection rifle or blowgun with lOmg/kg of Telazol Smith and Jungers (1997; Table 1). Also see Appendix (Fort Dodge Laboratories, Inc., Ft. Dodge, Iowa; Fig¬ la, the online Lepilemur Field Data Appendix, for all ure I; Appendix II). We recorded the location of all measurements and e-voucher photographs. immobilized lemurs using a global positioning system (Appendix la). Four 2.0 mm biopsies and 1.0 cc per Data Collection.—Genomic DNA was extracted kilogram of whole blood were collected from each from samples using a phenol-chloroform extraction and stored in room temperature storage buffer (Sambrook et al. 1989). From these samples, the fol¬ (Longmire et al. 1992). While immobilized, a Home- lowing regions of the mitochondrial DNA (mtDNA) Again microchip was placed subcutaneously between were amplified: the displacement loop or control the scapulas of each lemur (Appendix la). This proce¬ region (D-loop; Baker et al. 1993; Wyneret al. 1999); dure was used to field catalog each animal with a unique the 12s subunit ribosomal RNA gene, along with a recognition code in order to positively re-identify all portion of the tRNAplK' (12s rRNA; Hedges 1994; captured individuals during any future immobilizations. Wyner et al. 1999); and a fragment of the cytochrome In addition, morphometric measurements were taken. oxidase subunit 111 gene (COIII); NADH-dehydroge- Louis, Jr. et al.— Analysis of the Sportive Lemurs 3

Sahafary N Ankarana. Mahavavy Nord- ^.-Analamcra --'-Andrafiamena Lokobe.^ Sambirano«- 0_100 Manongarivo-.T km 1 Antafondro- Sahamalaza- Maevarano- Anj iamangirana —/ Sofia- "Anjanaharibe Sud Mahajamba ■Antainambaluna Betsiboka Anjahamena .Mananara Nord Ankarafantsika Tsiombikibo.^ Mahavavy Sud Maningoza Maningory .Zahamena Manambaho -Anjozorobe ■—Betampona Tsingy de Bemaraha Mantadia -Maromizaha Manambolo .-Anosibc an’ala Tsiribihina' ■Tsinjoarivo ^-Mangoro Beroboka Morondava

Mangoky Namorona Faraony

Zombitse'

Fihcrenana Manampalrana Onilahv Manombo

Beza-Mahafaly Mananara

Manangotry Mandrare •*.Collection Site MenamJdfHj 'Mangatsiaka <-River Ihazofotsy

Figure 1. Sample distribution map of the sportive lemurs of Madagascar. 4 Special Publications, Museum of Texas Tech University

Table /. Morphometric data collected from sedated individuals. (Individual morphological data available online; see Appendix la).

Species Name Common Name N Weight Head Body Tail Crown Length Length (kg) (cm) (cm) (cm)

Lepilemur aeeclis Aeecl’s Sportive Lemur 8 0.86±0.17 7.7+1.2 22.4+1.1 25.4+2.1 Lepilemur ahmansoni Ahmanson’s Sportive Lemur 4 0.61 ±0,15 6.2+ 1.2 20.6+1.6 23.8*0.7 L ep i lemur ankaranem is Ankarana Sportive Lemur 24 0.79±0.12 7.1 ±0.9 21.8+2.0 27.3+ 1.9 Lepilemur betsileo Betsileo Sportive Lemur 3 1.15±0.13 7.5±0.3 25.2+0.1 27.7+1.3 Lepilemur dorsalis Grey-Backed Sportive Lemur 6 0.73+0.07 7.3±0.4 22.8+1.5 27.5+0.9 Lepilemur edwardsi Milne-Edwards’s Sportive Lemur 11 U 0+0.11 6.7±0.6 26.3+2.7 28.5+2.1 Lepilemurfleuretae Fleurete’s Sportive Lemur 3 0.98+0.16 7.4±0.3 25.5+2.2 30.2+2.1 Lepilemur grewcocki Grewcock's Sportive Lemur 3 0.78+0.20 6.2±0.1 24.8+2.1 28.5+1.8 Lepilemur hubbardi Hubbard’s Sportive Lemur in 0.99+0.15 7.54122 23.4+1.4 24.0+1.1 Lepilemur James i James’ Sportive Lemur 2 0.78±2.76 8.0±0.3 25.7+1.5 29.7+1.8 Lepilemur leucopus White-Footed Sportive Lemur 18 0.54+0.09 6.5:07 19.6+1.4 24.2+1.2 Lepilemur microdon Small-Toothed Sportive Lemur 7 1.19+0.42 10.8±2.3 27.0+2.2 26.8+3.1 Lepilemur milanoii Daraina or Swimming Sportive Lemur 14 0.72+0.10 6.4±0.5 21.6+1.7 26.0+1.4 Lepilem ur m us tel inns Weasel Sportive Lemur 27 0.99+0.21 7.9±1.4 25.8±4.0 25.2+2.1 Lepilemur petteri Petter’s Sportive Lemur 3 0.6344X05 5.1 ±0.4 23.2±0.9 23.7+1.4 Lepilemur randrianasoli Randrianasoli’s Sportive Lemur 3 0.92+0.08 8.2±0.6 24.1±l .7 27.4+1.8 Lepilemur ruficaudatus Red-Tailed Sportive Lemur 3 0.86+0.06 6.9±1.0 24.3+3.6 24.2+1.0 Lep il em ur sahama tazensis Sahamalaza’s Sportive Lemur 2 0.70+0.07 6.3±0.1 19.6±0.3 23.7+2.7 Lepilemur seali Seal’s Sportive Lemur 6 0.95±0.08 7.4+0.9 27.3+1.4 26.3+1.4 Lepilemur septentrional is 2 0.58:0.18 8.3 U.l 18.7+1.7 24.8+4.5 L epilemur tymerku hson i Hawk’s Sportive Lemur 10 0.88±0.10 6.5+0.2 23.1 + 1.5 24.7+2.3 Lepilemur wrighti Wright’s Sportive Lemur 5 0.95+0.49 8.0± 1.3 25.2+3.1 25.5+1.6

nase subunits 3, 4L, and 4 (ND3, ND4L, and ND4); products from low quantity), the sequence data was as well as the tRNA^'fi tRNAAlN tRNAHis, tRNAScr, generated for the D-loop fragments, and 12s rRNA and and partial tRNAu*u genes (subsequently referred to PAST sequence generated from overlapping segments as the PAST fragment; Pastorini et al. 2000). Using were confirmed. The samples were electrophoresed 50 nanograms of genomic DNA, the D-loop (555 bp), on a 1.2% agarose gel to verify the PCR product and 12s rRNA (877 bp), and the PAST (2378 bp) fragments purified using QIAquick PCR purification kit (Qiagen; were amplified using the following conditions: 94°C Valencia, California). for 30s; 47°C for 1 min; 72°C for 5 min for 35 cycles. Since potential nuclear insertions or mitochondrial Using the BigDye terminator cycle sequencing pseudogenes within the nuclear genome can be ampli¬ ready reaction kit by Applied Biosystems the sequence fied inadvertently, we chose to minimize this likeli¬ was then run on a 7% polyacrylamide gel by an ABI hood by amplifying both mitochondrial DNA regions 377 automated sequencer (Applied Biosystems, Inc.; as intersecting or overlapping segments (Zhang and Foster City, California). Four published internal Hewitt 1996). Consequently, the 12s rRNA fragment sequencing primers, 12L1, I2L5, 12H2, and I2H3 was generated from two amplified segments, and the (Hedges 1994), w^ere utilized to generate the 12sRN A PAST fragment was generated from seven amplified sequence data, and a suite of internal sequencing segments. Additionally, to further eliminate amplifica¬ primers from Pastorini et al. (2000) and Pastorini et tion of nuclear insertions, a technique that is species al. (2001) were used to generate the PAST fragment. independent and both rapid and efficient derived from Additionally, PCR and sequencing primers specific for the degenerate oligonucleotide-primed PCR method Lepilemur were designed for the PAST fragment, and (DOP-PCR; Telenius et al. 1992) was used to generate PCR and sequencing primers specific for lemurs were the PCR products. Adapting this LL-DOP-PCR (long designed for the 12s rRNA and D-Loop fragments Louis, Jr. et al.— Analysis of the Sportive Lemurs 5

(Appendix Jc). The sequence fragments were aligned Bayesians inference analyses were conducted to generate a consensus sequence using Sequencher using MrBayes 3.0b4 (Huelsenbeck and Ronquist (Gene Corp; Ann Arbor, Michigan), and the consensus 2001; Ronquist and Huelsenbeck 2003). The model sequences were aligned using ClustalX (Thompson et of evolution was selected by using MrModeltest 2.2, al. 1997). All aligned sequences are available from a modified version of Modeltest 3.6 (Nylander 2004; the first author upon request. All sequences have Posada and Crandall 1998). A Markov Chain Monte been deposited in GenBank and the sequence data Carlo (MCMC) run with four simultaneous chains and and information are available from the referenced 1,000,000 generations was performed. The pattern accession numbers (Appendix II). of sequence evolution was estimated by conducting a minimum spanning network generated with the pro¬ Phylogenetic Analysis.—Maximum-parsimony gram NETWORK version 4.11 (Bandelt et al. 1999; analysis (MP) was performed for the phylogenetic Forster et al. 2001; Gonzales et al. 1998) and Arlequin, study of the D-loop, 12s rRNA fragment, PAST frag¬ version 2.0 (Schneider et al. 2000). We also conducted ment, and combined (12s rRNA and PAST fragments) Message Passing Clustering (MPC) (Geng et al. 2004, sequence data with PAUP* Version 4.0b 10 software 2005). MPC is an agglomerative, object-oriented (Swofford 2001). Heuristic searches were completed clustering algorithm that preserves the clustering pro¬ using the random addition sequence (1000 replicates) cess in a tree structure by employing the concept of with the tree bisection-reconnection branch swapping message passing to describe parallel and spontaneous routine. Gaps were considered as a fifth character in biological processes, such as speciation. The Kimura MP analyses, but were treated as missing data in the (1980) two-parameter nucleotide distances of the 211 neighbor-joining analyses (NJ). Bootstrap analyses Lepilemur samples were used as input for the MPC were accomplished with 1500, 1000, 2500, and 1000 analysis. These distances were generated using PAUP* pseudoreplicates with the D-loop, 12s rRNA, PAST, version 4.0b 10 (Swofford 2001). and combined data sets, respectively. Only nodes with greater than 50% support were reported. The maximum In addition to character-based phylogenetic anal¬ likelihood (ML) analyses were performed using the pro¬ ysis of DNA sequences, PAUP* software (Swofford gram DNAML in PHYLIP 3.65 package (Felsenstein 2001) was also used to calculate uncorrected pairwise 2005). Due to the large number of taxa and characters distances for 12s rRNA, ND3, ND4L, ND4, PAST and the resulting computational intensity, we pruned fragment, and 12s rRNA and PAST combined frag¬ the combined sequence dataset by choosing taxa from ments (‘p’). As described in Davis and Nixon (1992), distinct clades supported in an initial NJ (Saitou and Nei Louis et al. (2006), Mayor et al. (2004), and Wyner et 1987) analysis. The NJ analysis was performed with al. (1999), we utilized MacClade 3.01 (Maddison and MEGA 3.1 (Kumar et al. 1993) using Tamura and Nei Maddison 1992) and MEGA version 2.0 (Kumar et al. (1993) gamma distances with an a-parameter value - 1993) in a diagnostic search to designate evolutionarily 0.5, which was estimated using the Yang and Kumar significant units (ESU) using population aggregate (1996) method; the reliability of internal branches analysis (PAA) of the D-loop (544 bp), 12s rRNA (877 was assessed with 1,500 bootstrap pseudoreplicates. bp), and PAST (2378 bp) sequence data for Lepilemur. The taxa chosen from the NJ tree for the ML analysis With the sequential addition of each individual without included 55 taxa; RAN0229, a Greater an a priori species designation, a PAA distinguishes [Cheirogaleus major], was used as the outgroup taxon. attributes or apomorphic characters according to the The ML analysis of these taxa was performed using F84 smallest definable unit (Davis and Nixon 1992; Louis gamma distances with an a-parameter value = 0.5. The et al. 2006; Mayor et al. 2004). reliability of the internal branches was assessed with 1,000 bootstrap pseudoreplicates. 6 Special Publications, Museum of Texas Tech University

Results

Mitochondrial DNA sequence data was com¬ species (Figure 2). In order to verify that our samples pleted for three fragments, D-loop, 12s rRNA, and are indicative of the three newly described species in PAST fragment (approximately 3800 bp), for 211 in¬ Andriaholinirina et al. (2006), we used GenBank to dividuals, representing all eleven recognized species of BLAST cytochrome B subunit gene sequence gener¬ sportive lemurs from a total of forty-one sites (Figure ated for our data set. This confirmed that our samples 1; Appendix II). All new mtDN A sequences generated are representative of those species (GenBank Acces¬ for this study were deposited in GenBank and can be sion DQ529452-DQ529459; Andriaholinirina et al. acquired through the accession number (Appendix II). 2006). Additionally, 11 distinct subpopulations were The sequence alignments for the data sets are available identified within the currently recognized Lepilemur from the first author upon request. The 12s rRNA species distribution. Each subpopulation is geographi¬ fragment consists of the 3’ end of the phenylalanine cally defined by rivers which isolate them by acting tRNA (22 bp) and a partial section of the 12s rRNA as barriers to the recognized species. The results subunit gene (855 bp; Appendix Ig). The PAST frag¬ from the population aggregate analysis of the D-loop, ment consists of the 3’ end of the COIII gene (30 bp), 12s rRNA, and PAST sequence data are presented in the complete NADH-dehydrogenase subunits ND3 Tables 2A-C and 3A-C, respectively (the complete (348 bp), ND4L (297 bp), and ND4 (1378 bp), along diagnosis from the PAA for the D-loop, 12s rRNA, with the tRNA genes, glycine (73 bp), arginine (73 bp), and PAST sequence data are available online; see Ap¬ histidine (70 bp), serine (65 bp), and the 5’ portion of pendix Ie). Multiple diagnostic characters define each leucine (47 bp; Appendix Ig). The polyadenylation of Lepilemur species, along w ith the 11 newly described COIII and ND4 genes, insertion of base pairs between species (Tables 2 and 3). A review of the morpho¬ ingroup/outgroup comparisons, and other alignment metric data for the 22 species of sportive lemurs is characteristics between lemurs and Homo are consistent presented in Table 1 (detailed morphological with Pastorini et al. (2000). measurements of the individual are available online; see Appendix la). The complete un¬ Based on the phylogenetic inferences of the NJ, corrected lp’ distance and the Kimura two-parameter MP, and ML analyses of four sequence alignments distance measures are presented in Appendix Id. (D-loop, 12s rRNA, PAST, and 12sRNA-PAST frag¬ ment combined), three major Lepilemur subgroups The MP, Bayesian, and MPC analyses are pre¬ are represented, differentiating the eleven recognized sented in Figures 2 and 3 (also see Appendices Ih and sportive lemur species (Figures 2-4, 6-7; additional Ii). All analyses differentiate 22 Lepilemur species, figures are available at http://www.omahazoo.com). including 11 new species. The minimum spanning net¬ The first subgroup corresponds to the eastern sportive work presents diagrammatically the speciation among lemurs, L. mustelinus and L. microdon (the distribution the 22 sportive lemurs (Figure 5). The ML tree was of L. mustelinus and L. microdon is based on Andriaho- estimated from the combined 12s rRNA/PAST align¬ linirina et al. (2006) and Mittermeier et al. (2006); Ap¬ ment from a subset representing the haplotypes within pendix lb). The second subgroup includes the western the data set, and corresponds to the other phylogenetic and southern sportive lemurs, L. edwardsi, L. aeeclis, inferred trees (Figure 7). There is high bootstrap sup¬ L. randrianasoli, L. ruftcaudatus; and L. leucopus, port for the ML analysis with respect to the topology of respectively. The remaining subgroup consists of the the genera and species. Specific resolution of sportive recognized northern sportive lemurs, L. septentrional is, lemurs based on phylogenetic inference parallels geo¬ L. ankaranensis, L. dorsalis, and L. sahamalazensis. graphic separation into western, eastern, and northern There is high bootstrap support for the MP and NJ groups with the exception of L. microdon which is analysis with respect to the topology of the genera and aligned with the western group.

(text continued on page 18) Louis, Jr.etal.—AnalysisoftheSportiveLemurs Figure 2.Neighbor-joiningphylogramderivedfomD-loop DNAsequencedatafrom the distributionincurrent literature(Andriaholinirinaetal.2006;Mittermeier nodes. Valuesbelowbranches indicatesupportofbootstrappseudoreplicates.Length 67 haplotypesfromthe211Lepilemur individuals.Speciesdesignatedaccordingto = 1,698;Cl0.4346;RI-0.8299; RC=0.3607;HI0.5811. al. 2006;Appendixlb).Values abovebranchesindicatenumberofchangesbetween Branch to Outgroups MV/ -10 changes 192)' ' (West ofMenagnaraRiver) Lepilemur microdon —Lepilemur microdon Lepilemur microdon (East ofMandrareRiverandsouthMenagnaraRiver) Lepilemur microdon Lepilemur mustelinus (North ofManingoryRiver) (North ofNamoronaRiver) FIA5.11 ANAL2.1 2 ANAL13 98 CAR21 „„ 591I-IA5. Lepilemur edwardsi (North ofSofiaRiver) M iFIA5.1 j CAR6 Lepilemur mustelinus Lepilemur microdon (South ofManampatranaRiver) Lepilemur dorsalis Lepilemur leucopus (West oftheLintaRiver) Lepilemur leucopus Lepilemur septentrionalis Lepilemur sahamalazensis Lepilemur aeeclis (South ofMahavavySudRiver) Lepilemur randrianasoli Lepilemur aeeclis -Lepilemur rujicaudatus _Lepilemur ankaranensis (Nosy BeIsland) Lepilemur dorsalis Lepilemur ankaranensis (South ofMangokyRiver) — Lepilemurrujicaudatus (South ofLokyRiver) 7 8 Special Publications, Museum of Texas Tech University

-ANK7W— MiLiotvhiis tlfltJn l. -RANO250W-- Mivnx-cbn.s rufus - GAR8M— Chciroguknis mechus -//- -RAN022<34" Cluinigcileus fimi'ir .RAN067 L . . ,

_ ANK3.T4” .tviih ticiidenlalis 42 - MOR644- ProfHtkevm renvaim -BAN0331*" Propilheciix edwcinlxi

^ RAND35T I*" »W 24 Lucian j 24 i ,, . ->»AN0338f'“//t'P('/t'""" v""1 23 15 -ANAL2.23 UapttlenI mmtrg. occuienltdis 58 ^ - CrAK<; I 13 JRAN061. kNC)6ll „ , . It) 1RAN062 r nmtimw g - R AN045K—£/(/t//;,|(|- fuhtis rufus IAN2 IK legahl vttriei’UUl

F1AS.1 F1A5.12 CAR21 LepilvmuP imkaranensis ANAL2.1 ANAL13 FIA5.I1

Lepilenwr milanoii

Lepilenwr lyiiicrlai ■hsunt

3r ANT5.2 In n.‘ANT5.5 Lepilemw dorsalis r-—-I ANT5.11 UANT5.7 Lepilemw saliamulazensis

Lepilenwr ahnuwitnn

Lepilenwr septentrmmdis l ■ ZOMB8 2 f ZOMB12 Lepilemw huhbardi

Lepilenwr nificaudcitu.s

/.epi lemur ramirianasoli

Lepilenwr aeeclis

■AN 065 P 1|aND68 Lepilenwr leucopus AND70 BEZ14 Lepilenwr petteri

Lepilenwr mit radon

Lepilenwr edwardsi

HIH2I Lepilenwrgrewcockl ANJZ32 lj TA025 mjANOSIBIS "M1ZA4.18 .1S1N26 Lepilenwr nmstelnms BET21

Lepilenwr hetsileo

Lepilenwr /ame.si AMBioj Lepilenwr fleitrehte 20 ——-JAR2 , 21 mada.ti th Lepilemw seah

. KLALA4.18 ] Lepilenwr nriehti -10 changes L'kALA24 ‘

Figure 3. Phylogeny derived from D-loop fragment sequence data from 67 Lepilenwr haplotypes (one of 80 most parsimonious trees). Values above branches indicate number of changes between nodes. Lours, Jr. et al.— Analysis of the Sportive Lemurs 9

- ANK7 Microcebus ravelobensis RAN025Q Microcebus nifus Jq— GAR8 Cheirogalem medius '-RAN0229Cheirogaleus major

"I RAN03 381 ^aPalcm!r sunus

—-IRAN0352aWew JJ^RANO&IHllPalmur S- grisem •**** | Hapalemur g. occidentals

PANIWarecia v, variegata R AN045£w/cm urfulvus nijits LL-RA N03 32 Propithecus edwardsi 1 - MOR68^;fe«' verreauxi RAN026ll ^va^1 (viHgfr ANK33 Avahi occidentals

Lepilem ur ankaranensis Lepilemur milanoii Lepilemur alimansoni — Lepilemur sahamalazensis ^NTC-2 ; Lepilemur dorsalis Lepilemur tymerlachsoni _ Lepilemur septentrional is MARuJ Lepilemur edwardsi HIH21 <-Lepilemur edwardsi Lepilemur microdon ||BEZ14 , |-h BEZ18 Lepilemurpetten J 3 L BEZ21 AAND67 5 Lepilemur leucopus

Lepilemur ruficaudatus

Lepilemur hubbardi —Lepilemur randrianasoli

Lepilemur aeeclis

Lepilemur mustelinus ^ Lepilemur betsileo

Lepilemur jamesi

Lepilemur mustelinus

Lepilemur fleuretae

iHLKALA4.16 Lepilemur wrighti 11KALA4.9 >JAR2 1JAR3 Lepilemur seali NARA4.20

Figure 4. Phylogeny derived from combined 12s rRNA fragment sequence data from 54 haplotypes from 211 Lepilemur individuals (one of 128 most parsimoni¬ ous trees). Values above branches indicate number of changes between nodes. Length 1,080; Cl - 0.5 139; RI = 0.8629; RC = 0.4434; HI - 0.5132. 10

L.ankarcmensis ; A L.randrianasoli L milanoii ^ L.aeedis L.ivmerlachsoni L.hubbardi L. dorsalis ^ L.ruficaudaius Special Publications,MuseumofTexasTechUniversity

Figure 5. Minimum spanning network of Lepilemur haplotypes calculated using Arlequin Version 2 and Network Version 4.11. Identification numbers denote haplotypes corresponding to Appendix II. The minimum number of mutational steps separating matriarchal lines is indicated. Nucleotide substitutions are indicated by dashes. The number of nucleotide differences in their connecting lines (more than 10) is indicated when they are more than one. Missing intermediates are indicated by gray circles. The size of circles approximates the number of individuals with matching haplotypes (circles without any number represent one individual). Louis, Jr. et al.— Analysis of the Sportive Lemurs 11

Lepilemur ankaranensis

Lepilemur milanoii

Lepilemur tymerlachsoni

Lepilemur ahmansoni o Lepilemur sahamalazensis L ep 11 em u r dorsalis Lepilemur septentrionalis

^Lepilemur edwardsi

|Lepilemur grewcocki

iLepilemur microdon

Lbezu Lepilemur petteri

Lepilemur leucopus

[Lepilemur ruficaudatus

Lepilemur hubbardi }^epilemur randrianasoli

Lepilemur aeeclis

Lepilemur mustelinus

Lepilemur betsileo

Lepilemur jamesi

Lepilemur fleuretae Lepilemur wrighti

Lepilemur seali

Figure 6. Neighbor-joining phylogram derived from PAST fragment sequence data from 211 Lepilemur individuals, representing 156 haplotypes (the phylogram presented maintains branch lengths proportional to the number of changes). 12 Special Publications, Museum of Texas Tech University

MER26 ANAL2.29 CAR6 CAR45 MER24 ANAL5 ANAL2.4 Lepilemur ankaranensis LAME5.6 FIA5.12 FIA5.6 LABE5.2 FIA5.11

Lepilemur mil anoii

Lepilemur tymerlachsor Lepilemur sahamalazen, Lepilemur ahmansoni

Lepilemur dorsalis

Lepilemur septantnonai 968

Lepilemur edwardsi

Lepilemur grewcovki

Lepilemur mierodon

989 Lepilemur aeeclis

Lepilemur randrianasol

Lepilemur hubbardi MORI 17 Lepilemur ruficuudatus AND93 HAZ05.17 Lepilemur leucopus AND65 BEZ15 Lepilemur petteri BEZ21

[252 ANOSIB15 MIZA15 TAD4.23

TAD25 Lepi I em ur m us tel in us SIN27 ANJZ32 ■ ZAH25 li000TSINJ36

1000 Lepilemur jamesi

Lepilemur betsileo Lepilemur fleuretae Lepilemur wrighti 1000 JTooo NARA4.20 Lepilemur seali JAR3 RAN0229 Cheirgaleus major

Figure 7. Maximum-likelihood phylogram derived from combined 12s rRNAand PAST fragment sequence data from 55 Lepilemur individuals. The phylogram presented with support of 1000 bootstrap pseudoreplicates (values specified on the nodes). Louis, Jr. et al.— Analysis of the Sportive Lemurs 13

Table 2A. Diagnostic nucleotide sites from the D-loop Pairwise Aggregate Analysis (PAA) o/Lepilemur.

111 i 11 111 ] 1 1111 til I n 12 2 2 2Z2.2 2 2 2 2 2 3 3 3 3 3 3 3 3 1 4 4 5 5 5 5

1 1 122 3 3.33 34 4 44 55 555 Jfjjo 1.1 1.112252 2 2 / 3 3 3 3 3 9 9 1 1 4 4 567?.&8S99fr£l ]'3#8 9l 9 3 3 3 5

1 0 8 9 1 5 2 3 4 7 834 6 7 04 57 8 9 34^0 34 7 90 1 34 5 6 91 4 5 5838 280 7 1 80 1 4 5 690 J 8255 3 3 4 4 1 3 4 5 2

ANAL2. 1 CTCAACGCTATTAAA-AACCTTCCTGGAGC-ACCCAATTGCAACAfTTTTAAATCACC'.TCAAAACT

DAR 4.7 .T... , T . . .-G.

L0K04.7 .T... . T

FRAY 3 . f) 1 .T.C.G.C-CC--.T . .A ...... G . . . . A .

ANT 5.2 .TA... . T . . A . . . T -_ . . r: T A GA

LAZA5. 1 .TA . C.C-T- - . . . . . A A 0 . -.C . . T_ . T . . A. T

BEZ14 .A . . . .-C--T . . . . A . . . T-T.TTT . . . . T . , .A.

...G..C - - T . . . . AA . . T-T.TT . . . . T . . . G ,

MORI IT . . A. . .GGC. . .GG. . .TCC . . GCTA. . A . GC . .

Z0MB8 . . . A ...-C...GG....GTTG .. c. . GTC . . . G . . T . .

BEMA5 .AT. .A-C-C--G. . . . ,T . . AA . . ,CC. . .GG. . . .CT. . A . C . . . .C. . .G.

MAP 1 .A . . .CAT. .C-C--. . . . . C . r a .AGC-...CCC. . . T . . . A .

HIH21 .A. . .CAT. .C-C--. . . . . C . . . A . .AGC- . . .CTG. . . C . . . . T . . . T.

KIB02 2 TA . . ,C-T-- . . . . . AAGA ...... C. . . T .

JAM 4.27 . . . .G.A.C. .-C-C-- . . . r . . A. . . .-T, . .GG. . . .T. . . .AC. . G.T. . .A.

TAD2 5 .TAAC.CA.C.AACCA..T .AAC.GG...CA.TCT.. . . A . . .C. . ..ATCAC. , T . .

M8 3B .TAAC.CA.C.AACCA..T . C . . .AAC.AG. . .CA. .TT. . . . A. . .C. . T . A . .AC . .T. .

FAN! 0 .T. AC*ipwC, AACCA. . T r .AAC.GA..GCA.TCT.. CCG . . .c. . . . A . . AC . . T. .

AND2 0 .C_AA«A.CGAACCA. . . . C . . .AGC,®. . .C - . . .T. . . .ACG . C . . AGA. . AC.. . T . .

RAM02 3 4 .AA.G.CAC..C-CT1...1 TG.T -C. . . T . . . A.

KALA24 .TAA. .CA.C.TCACCG . T . . C . r TAA-.GGT. . .A.TT. . . . . C . . TC . . T . G . . ATC ■c-

JAR 2 T.T. . .TAC.CA.C.AGCCT . .A . .C . .0.. T.GT.GGT . .CAAACT . . . . A . . _ . . A. .AC . . T . . 14 Special Publications, Museum of Texas Tech University

Table 2B. Diagnostic nucleotide sites from the 12s rRNA Pairwise Aggregate Analysis (PAA) o/Lepilemur.

1 1 12 2 2 2 2 |f ftf3 314 4«4'44 5 8jf|6 6 667 7 7?17 17 7 8

1366 1 45 3 44 T-f J 3 4 § f f 113- J b 8 gtj 1.6 8 S 2 4' 5 5 6 6 $ 8 9 5

6 8 3 0 6 0 8 4 5 0 2 0 4 0 4 0 9 9 91 4 3 6 2 3 9 8 8 0 b 6 9 1 6- 4 3 3 6 0 7 1 6 8 6- 6 4

ANAL5 A A G A C T C T A A C A A G A A T C. C C Gr 1AAAATATAAAC AACAACACCCGGT

DAR4.4 .G . . 1' .T . . ,

L0K04.2 .A , . . T . . . . I.r

FAR75.1 . G . ! 3 . G . C.G. , , , C . G .

ANT5 . 2 . .T. . .T...A.

LAZA5.1 .G. . . T . . .T . . . .

BEZ14 .G.T ...... C . G .

AND 6 5 T T

MGR117 ■T' f;

Z0MB8

BEMA5 T . . .T.A . T . . c. .

MARI .. c. . . , . T . .

H 1 H 2 1 . . r . . . . . T . .

KIB022 . . T . T G T G

JAM4.27 r g

TAD25 C . . G r; , T . . . , c T A

M83B C . . . . TC. . .C .A . . . T . . . . , . C T A

FAN 10 c. . , .C.

AND20 C . A .

RAN0234 . . T . G . . G.

KALA24 C . . . ■ C.A.

JAR2 GT. . Table 2C. Diagnostic nucleotide sites from the PAST Pairwise Aggregate Analysis (PAA) o/'Lepilemur. Louis, Jr.etal.—AnalysisoftheSportiveLemurs io r' i OGAYO 1 r-lono I hooo CM in r-yo YD HiD YD CMOA YD vDGO yo p Y0 00H co ono •GO YO ■ IJ&S“sP C\l CMO no oaoo YT OvO O’ ^03 m OlO in rHO lo no LO novt lo no oo no J1 nom >n JS r-oa CO 04vp 00 CMOA oo o' 00 lO o m o oar- •—I O1-0 —I rH I OA oa o on oo no CO ‘M* GO LO O to LO in YO yd OA r- o yo yC OA n& 3*^3*f-Ci -•NOtfiMlflTHNOfl; CM < M-H £

mill 111111111 11 il 11114X1113 UltljXIIl 14ll;tllll11 llllXi'U 1114x11 milllllll 2222222222222222222Z222:2M22 llll421422223333333333333341444444 444&555'55$5&6«l??77788888e-89t393999999i:990aoaO,D4‘llltll;llil.llll4t22.233 22 772233478 903444 5556-677M02234445573Q2334537613037333Ql3p3€9l002335S78899912P5Gt90i 4445667777SB 3345936 041468l,50H54923784788S'O8'6'8213.t248 3640754 78Of4330837O3348576l€8871894Q9l-g©3 5Ml;68'9€7ll46338'07'89l50€-5l448 . 'If •*■••• ip O «j= H P iU St o u p t > p 0 CJ u 0 0 P o P «1 R t-* 0 a) u 0 p p u p u Si 0 0 0 < lo t; / ■p 0 fSC P p p| P u 0 p P e u u 0 P p u < 2 O (J u u p u u 0 p u a § P (4 rf! P P O P u P Si p u < ) 0 to s () to 1 I w u Pi 00 to R to to P CM 0 (') m U 1 TR Lh H m LO to H to p to w ■si P () P 1 1 H .—1 to n] to to P 1 1 Special Publications,MuseumofTexasTechUniversity :<■ H -MJ to a ;r( H to 1 H :f^4 ' M to CM H to to p i 1 P :H P Ol (to P l- ( 'i (J R H 1 4 1 R lO r> U p u R Q p P 1 1 CJ p —1 00 m O c : o to O < O to P >2 i pC P Pi c D r) t' r) il-l ,—l to to () •: > P P l 1 to 0 O P !-■ CM to to ( 1 r< Z rj ! J p- a p 1 I <] F~f CM P P 0 to 0 rx p to —1 1 1 R P

Table 3A. Summary of Population Aggregate Analysis (PA A) D-Loop diagnostic sites for Lepilemur species. Refer to Table 2A. *No Character or Attribute is available for this fragment.

Species Fragment Size (bp) PAA base pair location

Lepilemur ankaranensis 540 * L epilemur rn ilanoii 540 129 Lepilemur lymerlachsoni 538 117, 301 Lepilemur septentrional is 536 43,46, 99, 113, 114, 247 Lepilemur dorsalis 540 534,535 Lepilemur sahamalazensis 542 * Lepilemur petteri 534 * L ep He mur leuc op i is 535 19 Lepilemur ruficaudatus 535 103, 126. 247,308,533 Lepilemur hubbardi 535 240, 251,268,300 Lepilemur randrianasoli 538 33, 38, 270 Lepilemur edwardsi 545-546 137 L epil emur gre wcoeki 544 137, 193,355 Lepilemur ahmansoni 542 * Lepilemur aeeelis 537-538 21,271 L ep i l em ur must elm us 552-553 387, 388 L ep il emur James i 552 131 Lepilemur betsileo 553 134, 270, 271,284 Lepilemur fleuretae 550 10, 37,47, 198, 285, 286, 312, 315 Lepilemur mierodon 530 25,34,44,57, 107, 110, 119, 120, 121, 123. 124, 125, 135, 136, 137, 394,552 Lepilemur wrighti 551 50, 54, 55, 58,299, 474,491 Lepilemur seah 550 1, 18, 32, 54, 104, 126,212,218,300

Table SB. Summary of Population Aggregate Analysis (PAA) 12s rRNA diagnostic sites for Lepilemur species. Refer to Table 2B. *No Character or Attribute is available for this fragment.

Species Fragment Size (bp) PAA base pair location

L epil emur ankaran ens is 855 * Lepilemur milanoii 855 498 Lepilemur tvmerlachsom 855 394,693,854 L ep i lemur septentrional is 855 18, 60, 110, 154,334,433,488 Lepilemur dorsalis 855 761,796 Lepilemur sahamalazensis 855 294,766 Lepilemur petteri 855 240 Lepilemur feu copus 855 * Lepilemur ruficaudatus 855 433 L ep He mi ir ft u b hardi 855 340 Lepilemur randrianasoli 855 6, 242, 369, 391,418,666 L ep Hem ur edwards i 854 * Lepilemur greweoi ‘ki 854 * Lepilemur ahmansoni 855 413,641,723,750, 768 Lepilemur aeeelis 855 389, 556 L ep demur mustelin i is 854 * Lepilemur famesi 854 66 Lepilemur betsileo 853 434 L epilemur fleuretae 854 53, 500, 757 Lepilemur mierodon 855 148, 235, 270, 757, 786 Lepilemur wrighti 854 349. 746 Lepilemur seali 855 6,310, 469, 536, 579, 684 18 Special Publications, Museum of Texas Tech University

Table 3C. Summary.> of Population Aggregate Analysis (PAA) 12s rRNA diagnostic, sites for Lepilemur species. Refer to Table 2C. *No Character or Attribute is available for this fragment.

Species Fragment Size (bp) PAA base pair location

Lepilemur ankaranensis 2359-2360 364, 858, 1804 Lepilemur milanoii 2359 342, 769, 1896 Lepilemur tymerlachsoni 2359 152, 328, 1309, 1378, 1456, 1861, 1898. 1995 Lepilemur septentrionalis 2361 44, 113,211.214, 274,353,354, 533, 551,555,576. 674, 734, 1103, 1174,1231,1347,1399, 1448,1550,1603, 1777,2144,2146, 2366 Lepilemur dorsalis 2361 579,717,746. 1507, 1525, 1780,2163,2168,2177, 2236 Lepilemur saham al azensis 2360 399, 539, 737, 749, 770, 803, 1358 Lepilemur petteri 2360 337, 578, 779,957, 1615 Lepilemur leucopus 2360-2361 220, 448, 719, 836, 1960 Lepilemur ruficaudatus 2360 94, 127,365,667, 776,919, 1074, 1370, 1783, 1835. 1921,2068 Lepilemur habbardi 2361 350,543, 566, 629,681, 1015. 1240, 1396, 1559, 1906, 1907,2111, 2245 Lepilemur randrianasoli 2360 195,397,699, 849,923, 1018, 1035. 1053. 1432, 1444, 1753, 1981, 1988, 2251 Lepilemur edwardsi 2360 856. 1108, 1194, 1343, 1474, 1979 Lepilemur grewcocki 2360 244,274, 406,629, 888, 896, 988, 1114, 1226, 1354, 1537,2130 Lepilemur ahmansoni 2360 46. 304, 350, 1096, 1097, 1285, 1402, 1818,2141,2170 Lepilemur aeeclis 2360 535, 548, 563, 581, 761, 975, 1357, 1368, 1423, 1442, 1480, 1990, 2089, 2107 Lepilemur mustelimts 2359-2360 85 Lepilemur jamesi 2360 140. 716, 2144 Lepilemur betsileo 2360 8, 1057 Lepilemur fleuretae 2360 29,103.269,358, 533,534. 546, 553,664,905, 1124, 1574,2013, 2023 Lepilemur micradon 2361 146, 510, 581.596, 826, 829, 1171, 1369, 1708, 1954, 1991 Lepilemur wrighti 2360 55, 133,663,696, 886,871,907,942, 1105, 1117, 1120, 1294, 1837, 1856, 1936, 2041,2096 Lepilemur sea/i 2361 83, 84, 131, 166, 170. 187,331,409, 505,558,572,615,695, 853,917, 937, 1045, 1228, 1235, 1270, 1332, 1348, 1421, 1423, 1453. 1548, 1795, 1921, 1939, 2056,2153,2178,2179, 2296

Discussion

The persistent and rapid loss of habitat and the especially in primates (Pope 1996). Even if the loss resulting fragmentation of panmictic populations have of genetic diversity in primates is characterized by an compelled wildlife and conservation agencies to take overall loss of heterozygosity with most of the varia¬ protective action according to existing guidelines and tion found between populations and/or social groups, information with the ultimate goal of prioritizing spe¬ the genetic loss is dependent on the proportion of the cies and/or sites. The explosive rate of the deforestation gamete pool contributed by each generation and by in Madagascar, however, has eliminated many of the lineage effects (Pope 1996). The management of frag¬ available options. Because haplotypes can be unique mented populations is further complicated by a lack of to each population, simply preserving one population information, such as full extent of species distribution, will not necessarily maintain species-wide genetic vari¬ from existing populations, and by the mating system ability. The extinction of any remnant population may and social structure of each species. Therefore, it is not diminish the between-population variance, but may important to maintain the composite genetic diversity result in the permanent loss of non-neutral alleles from and the species reservoirs within and among these the composite gene pool. The loss can potentially have fragmented habitats. deleterious effects with the loss of genetic diversity Louis, Jr. et al.— Analysis of the Sportive Lemurs 19

Historically, sympatric reproductive isolation, as Type Locality.—MADAGASCAR, Prov¬ described in the Biological Species Concept (BSC), has ince de , Tsiombikibo Classified Forest, been the predominant defining criteria of species status 16°02'24.7"S, 045°48' 10.6"E, and northwest of the (Mayr 1942). Unfortunately, this concept is difficult . to implement or delineate when the putative species in question is a geographically isolated or allopatrically Description.—L. ahmansoni is a smaller sized defined population. In this paper, the current Lepilemur sportive lemur (0.61 kg) compared to L. aeeclis (0.86 taxonomy was examined according to the Phylogenetic kg) with a pelage that is primarily dark gray on the Species Concept (PSC) sensu Wheeler and Platnick et body and diffuse reddish-brown on the dorsal surface al. (2000), Louis et al. (2006), and Mayor et al. (2004). of the extremities, especially distally. They have similar The diagnostic characters or attributes define evolution- color patterns to L. aeeclis except L. ahmansoni lacks arily significant units (ESUs). Several authors suggest the prominent dorsal stripe on the dorsal midline of the that ESUs are equivalent to species as determined back as described for L. aeeclis (Andriaholinirina et through the Phylogenetic Species Concept (Amato et al. 2006). A diffuse black stripe can be present on the al. 1998; Barrowclough and Flesness 1996; Cracraft dorsum of the head. The ventrum is dark gray towards 1983). The identification of eleven new species in the the midline but diffuses to a light gray ventrolaterally. following descriptions establishes the essential need for The tail is primarily reddish brown, darker on the extensive as well as detailed sample collections across dorsal surface than the ventral portion which is a light Madagascar to determine geographic ranges for all of grayish blonde. the sportive lemurs. The constant addition of samples to the PAA data set will continue to test the distinction Diagnosis.—In the D-loop, 12s rRNA, and PAST of these characters. sequence fragments, L. ahmansoni differs from the closest relative, L. sahamalazensis, by 5.5%±1% (33 Due to the inability to maintain sportive lemurs informative sites), 1.6%±0.4% (13 informative sites), as long-term live vouchers in captivity, whole blood, and 2.7%±0.3% (70 informative sites), respectively. In morphometric, and e-voucher photos will serve as the the D-loop, 12s rRNA, and PAST sequence fragments, type series for several of the newly described species. L. ahmansoni differs from the closest species relative to In each case, an attempt was also made to identify ex¬ geographic distance, L. aeeclis, by 9.6%± 1.3% (69 in¬ isting museum specimens to represent the type series formative sites), 6.5%±0.8% (59 informative sites), and and was listed in that section. 11.7%±0.6% (282 informative sites), respectively.

Lepilemur ahmansoni, New Species Distribution.—L. ahmansoni is currently known from the Tsiombikibo region, northwest of the Maha¬ Type Series.—Whole blood for KIB022 vavy River. The southern extent of L. ahmansoni is (TK125529/TTU1 04462), adult male; KIB058 unknown. Further work needs to be conducted to de¬ (TK125530/TTU104463), adult female; and KIB065 termine if the Maningoza, Manambaho, or Manambolo (TK 12553 1/TTU104464), adult female; are stored River is the southern range of L. ahmansoni and the and curated at The Museum of Texas Tech University, northern range of L. randrianasoli. individual measurements, e-voucher photos, and col¬ lection data are available online (see Appendix la) and Comparisons and Remarks.—L. ahmansoni is from The Museum of Texas Tech University under the smaller in size than L. ruficaudatus, L. aeeclis, L. ran¬ respective TTU catalog numbers. KIB022, KIB058, drianasolir, and L. edwardsi (Table I). Both KIB058 and KIB065 were collected by Richard Randriamam- and KIB065 were adult females, supporting offspring. pionona, Jean C. Randriamanana, Gerard Nalanirina, Although L. ahmansoni is located geographically Rambinintsoa Andriantompohavana, and John R. Za- closer to L. aeeclis and L. edwardsi, L. ahmansoni is onariveloon 23 October 2003, 27 October 2003, and taxonomically closer to the northwest sportive lemurs, 29 October 2003, respectively. L. sahamalazensis and L. dorsalis (Figures 2-6; each species is depicted by specific color throughout the fig- 20 Special Publications, Museum of Texas Tech University

ures). Additional survey work is required to determine of dark to light gray and reddish brown fur, darker the southern range of L. ahmansoni and the northern dorsally than ventrally. The pelage is noticeably lighter extent of L. randrianasoli. The molecular analysis pre¬ within the ear pinna bordered by dark brown to black sented in this paper corroborates the phylogeographic fur along the outer edge of the anterior aspect of the study of Pastorini et al. (2003) and Andriaholinirina pinna. The anterior portion of the mandible is white et al. (2006), with the forming the in color with the rest of the face gray in color. The southern limit of L. edwardsi. Further studies should tail contrasts sharply with the rest of the body, being be conducted north of Ankarafantsika National Park to black in color. determine the northern extent of L. edwardsi. Diagnosis.—In the D-loop, 12s rRNA, and PAST Etymology.—The name ahmansoni is proposed in sequence fragments, L. betsileo differs from its clos¬ honor of Robert Ahmanson andtheAhmanson Founda¬ est relatives by both genetic and geographic distance, tion who have given extensive support to the graduate L. mustelinus and L. microdon, by 5.2%±0.9% (42 programs of the Malagasy students in Madagascar and informative sites) and 11.5%±1.3% (76 informative at the Henry Doorly Zoo’s Center for Conservation sites), 0.7%±0.2% (9 informative sites) and 6.8%±0.8% and Research. (60 informative sites), and 2.2%±0.2% (67 informa¬ tive sites) and 15.7%±0.8% (370 informative sites), Vernacular Names.—Ahmanson’s Sportive respectively. In the D-loop, 12s rRNA, and PAST Lemur. sequence fragments, L. betsileo differs from another new species, L. jamesi, by 5.8%±1% (34 informative Lepilemur betsileo, New Species sites), 1%±0.3% (8 informative sites), and 1.8%±0.2% (41 informative sites), respectively. Type Series.—Whole Blood for FAN 11 (TK 1255 13/TTU104446), adult female; and FAN4.24 Distrihution.^L. betsileo is currently known (TK 125514/TTU104447), adult male are stored and from the Fandriana region, between the Mangoro and curated at The Museum of Texas Tech University. Namorona Rivers. The southern and northern distribu¬ Individual measurements, e-voucher photos, and col¬ tion of L. betsileo is unknown and further work needs lection data are available online (see Appendix la) and to be conducted to confirm the extent of its range. from The Museum of Texas Tech University under the respective TTU catalog numbers. FAN 11 and FAN4.24 Comparisons and Remarks.—L. betsileo is in¬ were collected by Richard Randriamampionona, Jean termediate in size to L. mustelinus and L. microdon C. Randriamanana, Gerard Nalanirina, Rambinintsoa (Table 1). FAN11 was an adult female, supporting an Andriantompohavana, and John R. Zaonariveloon 28 offspring. L. betsileo is located geographically clos¬ October 2000 and 2 April 2004, respectively. Accord¬ est to L. microdon to the southeast and L. mustelinus ing to Jenkins (1987), a specimen, ZD. 1981.759 (skin to the north (Figures 2-6). Additional survey work is and skull), is held at the British Museum of Natural required to confirm the southern and northern ranges History from the Ambohimitombo Forest (20°43’S, of L. betsileo. The range of L. microdon appears to 047°26’E), which is located within the tentative range extend in a northeast to southwest trajectory from of L. betsileo. Ranomafana National Park to Andringitra National Park. The southern limit of L. betsileo most likely is Type Locality.— MADAGASCAR, Province the . de Fianarantsoa, Fandriana Classified Forest, (ap¬ proximately 20°23'40.5"S, 047°38'06.6"E), and on the Etymology. — The name betsileo is proposed for . this species and is derived from the Malagasy tribe from the Fianarantsoa region. Description.—This sportive lemur is relatively large (1.11 kg) with a predominantly grayish to reddish Vernacular Names.—Betsileo Sportive Lemur. brown color pattern. The overall pelage is a mixture Louis, Jr. et al.— Analysis of the Sportive Lemurs 21

Lepilemur fleuretae ^ New Species Distribution.—L. fleuretae is currently known from the Manangotry region, the rainforest parcel Type Series.—Whole blood for AND20 of Andohahela National Park between the Mandrare (TKI25559/TTU104492), adult male, is stored and River to the west and the to the north curated at The Museum of Texas Tech University. (Figure 8). The southern and northern distribution of Individual measurements, e-voucher photos, and col¬ L. fleuretae is confined to the rain forest within the lection data are available online (see Appendix la) and boundaries of the Mandrare and Mananara Rivers. from The Museum of Texas Tech University under the Further work needs to be conducted to confirm the TTU catalog number. AND20 was collected by Richard extent of its range. Randriamampionona, Jean C. Randriamanana, Gerard Nalanirina, and Edward Louis on 12 May 2004. Comparisons and Remarks.—L. fleuretae is ap¬ proximately the same size as L.jamesi, but is smaller Type Locality.—MADAGASCAR, Province de than L. mustelinus and L. wrighti (Table 1). L.fleuretae Toliary, Manangotry, Andohahela National Park (ap¬ is the most genetically divergent of all of the southeast¬ proximately 24°45'46.0"S, 046°51'47.0"E). ern Lepilemur species with the exception of L. wrighti and L. microdon (Figures 2-8). The type series of L. Description.—This sportive lemur is medium¬ microdon is located south of Fianarantsoa which sets sized (0.80 kg) with predominantly gray pelage, with the distribution of this recognized species at Rano- a grayish-brown mixture along the proximal portion of mafana National Park (Andriaholinirina et al. 2006; the extremities. The pelage is noticeably lighter over Petter et al. 1977; Figure 8). Genetically, L. fleuretae the eyelids from the rest of the face. Running along is within the L. mustelinus clade representing the east the midline, a diffus stripe is present, starting from the coast Lepilemur group, whereas L. microdon is closely forehead and continuing approximately halfway down related to L. edwardsi, an east coast sportive lemur the back. The venter is a lighter brownish gray with (Figures 2-7). Further studies should be conducted some light brown along the lateral edges of the belly. north of Manangotry to confirm the northern extent of The tail is reddish gray proximally, becoming darker L. fleuretae (Figure 8). gray towards the tip. Etymology.—The name fleuretae is proposed Diagnosis.—In the D-loop, 12s rRNA, and in honor of Madame Fleurete Andriantsilavo, former PAST sequence fragments, L. fleuretae differs from Secretarire General du M1NENVEF. Madame Fleurete its closest relative by genetic distance, L. mustelinus, worked tirelessly and constantly strove for the long by 7.7%±1.0% (59 informative sites), 1.5%±0.4% (19 term conservation of Madagascar’s protected and informative sites), and 4.5%±0.4% (177 informative unprotected areas. sites), respectively. In the D-loop, 12s rRNA, and PAST sequence fragments, L. fleuretae differs from its closest Vernacular Names.—Fleurete’s Sportive Lemur. relatives by both geographic and genetic distance from two other new species, L.jamesi and L. wrighti, by Lepilemur grewcocki. New Species 7.6%± 1.1% (44 informative sites) and 10.2%± 1.2% (70 informative sites), 1.9%±0.5% (18 informative sites) Type Series.—Whole blood for HIH21 and 3.1 %±0.5% (27 informative sites), and4.3%±0.7% (TK1 25 51 9/TTU1 04452), adult male; HIH22 (113 informative sites) and 7.6%±0.6% (187 informa¬ (TK125520/TTU104453), adult female; and HIH23 tive sites), respectively. In the D-loop, 12s rRNA, and (TK125521 /TTU 104454), adult female; are stored PAST sequence fragments, L. fleuretae differs from its and curated at The Museum of Texas Tech Univer¬ closest relative by geographic distance, L. Ieucopus, by sity. Individual measurements, e-voucher photos, and I4.1%±1.5% (92 informative sites), 7.0%±0.9% (62 collection data are available online (see Appendix informative sites), and 16.9%±0.8% (409 informative la) and from The Museum of Texas Tech University sites), respectively. under the respective TTU catalog numbers. HIH21, 22 Special Publications, Museum of Texas Tech University

km Bcmarivo

Mahavavy Sud

Anuiinambalana Maningoza Maningory Onibe

Manambaho

Mangoro

River

Namorona Faraony

t L.ankaranensis L.leuco/ws Fiherenana \ L.milanoii I L.randrianasoli -Manampairana ) L.tymerluchsoni \ L.aceclis Onilahv L.dorsalis L.hubbardi Mananara ) L.ahmansoni L.ruficaudatus L.sahamalazensis i L.seali ) /...v epten Irion a I is L. wright i I L.edwardsi y I L.jamesi Linta L.grewcocki I L.betsileo I L.microdon Mandrare L.mustelinus Mcnarandra L.petteri I f. fleuretae

Figure 8. Proposed distribution of the sportive lemurs of Madagascar. Louis, Jr. et al.— Analysis of the Sportive Lemurs 23

HIH21, HIH22, and HIH23 were collected by Richard Etymology.—The name grewcocki is proposed Randriamampionona, Jean C. Randriamanana, Gerard in honor of Bill and Bemiece Grewcock who have Nalanirina, Rambinintsoa Andriantompohavana, and generously supported our fieldwork in Madagascar John R. Zaonarivelo on 13 November 2004. and provided the laboratories and housing for all of the Malagasy graduate students in Omaha. The Type Locality.—MADAGASCAR, Province de Grewcock’s support is instrumental, transferring con¬ Mahajanga, Anjiamangirana Classified Forest (ap¬ servation related technology to the future Malagasy proximately 15°09'14.9"S, 047°43'41.0"E). scientists.

Description.—L. grewcocki is a medium-sized Vernacular Names.—Grewcock’s Sportive Lemur. sportive lemur (0.78 kg) with a predominantly gray color pattern. The area around the mandible and the Lepilemur hubbardi, New Species dorsal surface of the snout is whitish-pink in coloration. A dark stripe is present on the dorsal midline surface Type Series.—Whole blood for ZOMB9 of the head. This black stripe may continue dorsally (TK125558/TTU104491), adult female; ZOMBI2 onto the back, but this character is individually variable. (TK125556/TTU104489), adult male; and ZOMBI5 The venter is light gray to white. Unlike L. edwardsi, (TK125557/TTU104490), adult male; are stored and which has a consistently white tipped tail, the tail of curated at The Museum of Texas Tech University. L. grewcocki is entirely gray. Individual measurements, e-voucher photos, and col¬ lection data are available online (see Appendix la) Diagnosis.—In the D-loop, 12s rRNA, and and from The Museum of Texas Tech University PAST sequence fragments, L. grewcocki differs from under the respective TTU catalog numbers. ZOMB9, its closest relative by genetic distance, L. edwardsi, ZOMB12, and ZOMBI5 were collected by Richard by 5.3%±I.0% (35 informative sites), 0.8%±0.3% (8 Randriamampionona, Jean C. Randriamanana, Gerard informative sites), and 2.8%±0.3% (66 informative Nalanirina, and Rambinintsoa Andriantompohavana on sites), respectively, in the D-loop, 12s rRNA, and PAST 15 March 2004, 15 March 2003, and 16 March 2003, sequence fragments, L. grewcocki differs from its clos¬ respectively. est relatives by geographic distance, L. sahamalazensis, by 6.8%± 1.1% (51 informative sites), 4.8%±0.7% (40 Type Locality.—MADAGASCAR, Province informative sites), and 9.9%±0.6% (239 informative de Toliary, Zombitse National Park (approximately sites), respectively. 22°53T8.7"S, 044°4r43.3"E).

Distribution.—L. grewcocki currently is known Description.—L. hubbardi is a large-sized sport¬ from the Anjiamangirana region, south of the Maha- ive lemur (0.99 kg) with reddish-brown, gray, and jamba River and north of the Maevarano and Sofia white pelage. The face is grayish-brown around the Rivers. muzzle and eyes with a reddish-brown dorsal surface crown. The fur around the neck is lighter, forming a Comparisons and Remarks.—L. grewcocki (0.78 reddish blonde collar. The dorsum is dark reddish- kg) is smaller than L. edwardsi (1.10 kg), and larger than brown around the shoulders and upper back, gradually L. sahamalazensis (0.70 kg). Although L. grewcocki is becoming a lighter reddish-white to gray towards the located closer geographically to L. sahamalazensis, L. base of the tail and hips. The venter is entirely white. grewcocki is taxonomically more closely related to the The tail is uniformly reddish blonde. western sportive lemur, L. edwardsi, and southeastern sportive lemur, L. microdon (Figures 2-6). Further Diagnosis.—In the D-loop, 12s rRNA, and studies should be conducted north of Ankarafantsika PAST sequence fragments, L. hubbardi differs from National Park to determine the northern extent of L. its closest relative by geographic and genetic distance, edwardsi, and south of Anjiamangirana to determine L. ruficaudatus, by 6.4%±1% (41 informative sites), the southern extent of L. grewcocki (Figure 8). 1.4%±0.4% (15 informative sites), and 3.5%±0.3% (141 informative sites), respectively. 24 Special Publications, Museum of Texas Tech University

Distribution. - L. hubbardi is currently only along the jaw and throat from the chin to the ears. known from the Zombitse National Park region, The dorsum of the head is brown with a black mid line north of the and south of the Mangoky which is continuous for almost the entire length of the River. body. The dorsal region of the large cupped shaped ears is gray with the borders edged with black. There Comparisons and Remarks.—L. hubbardi (0.99 is usually a small cream colored patch restricted to the kg) is larger in size than L. ruficaudatus (0.86 kg). Two region beneath the ears. The venter is primarily brown, phenotypes have been seen in Zombitse National Park. but a lighter shade than the dorsal pelage. Grayish- Currently, the sample size is too small to categorically brown pelage is found around the ventral surface of determine if the phenotypes are sexually dichromatic. the extremities and belly. The tail is brown proximally, Additional survey work is required to determine the gradually becoming a darker brown to black distally. southern range of L. hubbardi and the northern extent The pelage is shorter than L.fieuretae and L. betsileo, of L. ruficaudatus (Figure 8). giving the smooth appearance to the coat.

Etymology.—The name hubbardi is proposed in Diagnosis.—In the D-loop, 12s rRNA, and PAST honor of Theodore F. and Claire M. Hubbard Family sequence fragments, L. jamesi differs from its closest Foundation for their generous support of Malagasy relatives (two newly described species) by geographic graduate students in the field and in the laboratory and genetic distance, L. betsileo and L. fieuretae, by at Henry Doorly Zoo’s Center for Conservation and 5.8%±l% (34 informative sites) and 7.6%±1.1% (44 Research (CCR). By providing the new laboratory informative sites), 1 %±0.3% (8 informative sites), and and housing for the CCR’s Genetics Department, the 1.8%±0.2% (41 informative sites) and 4.3%±0.7% (113 Hubbard Family Foundation’s support will continue informative sites), respectively. to be instrumental in transferring conservation related technology to the future Malagasy scientists. Distribution.—L. jamesi is currently known from Manombo Special Reserve region, south of the Manam- Vernacular Names.—Hubbard’s Sportive Lemur. patrana River and north of the Mananara River.

Lepilemur jamesi, New Species Comparisons and Remarks. —L. jamesi (0.78 kg) is approximately the same size as L. fieuretae (0.80 kg), Type Series.—Whole blood for M140 (TK 1.25538/ but L. jamesi is smaller than L. betsileo (1.11 kg). Addi¬ TTU104471), adult male; M14I (TK 125539 tional survey work is required to determine the precise TTU104472), adult male; and Ml42 (TK 125540/ southern and northern range of L. jamesi (Figure 8). TTU104473), adult male; are stored and curated at These two rivers already act as barriers for two other Museum of Texas Tech University. Individual measure¬ species, “pure” Eulemur albocollaris and a proposed ments, e-voucher photos, and collection data are avail¬ species of Mcrocebus (Louis et al. 2006; Mittermeier able online (see Appendix la) and from The Museum et al. 2006). L. jamesi is found in one of the few re¬ of Texas Tech University under the respective TTU maining low altitude, coastal, rain forests. catalog numbers. M140, M141, and M142 were col¬ lected by Richard Randriamampionona, Jean C. Ran- Etymology.—The name jamesi is proposed in driamanana, Gerard Nalanirina, John R. Zaonarivelo, honor of the Larry, Jeannette, and Barry James’ Family and Edward Louis on 13 November 2000. for their generous and long term support of Malagasy graduate students in the field and in the laboratory Type Locality.—MADAGASCAR, Province de at Henry Doorly Zoo’s Center for Conservation and Fianarantsoa, Manombo Special Reserve (approxi¬ Research. mately 23°01 '69.5"S, 047°43'84.1 "E). Vernacular Names.—James’ Sportive Lemur. Description.—L. jamesi is a large sized sport¬ ive lemur with primarily brown pelage. The face is demarcated into a mask, with whitish gray marking Lours, Jr. et al.— Analysis of the Sportive Lemurs 25

Lepilemur mil a noil. New Species by 2.7%±0.6% (20 informative sites), 5.9%±1% (38 informative sites), and 5.1%±0.9% (39 informative Type Series.— Whole blood for DAR4.7 sites); 1.9%±0.4% (18 informative sites), 4.4%±0.6% (TK1255 16/TTU 104449), adult female; DAR4.17 (38 informative sites), and 2.9%±0.5% (25 informa¬ (TK125517/TTU104450), adult male; and DAR4.18 tive sites); and 1,2%±0.2% (33 informative sites), (TK 125564/TTU 104497), adult female; are stored 8.4%±0.5% (198 informative sites), and 4.2%±0.3% and curated at The Museum of Texas Tech University. (100 informative sites), respectively. In the D-loop, Individual measurements, e-voucher photos, and col¬ 12s rRNA, and PAST sequence fragments, L. milanoii lection data are available online (see Appendix la) and differs from another species that is geographically from The Museum of Texas Tech University under the close and newly described, L. tymerlachsoni, by a respective TTU catalog numbers. DAR4.7, DAR4.17, genetic distance of2.5%±0.6% (18 informative sites), and DAR4.18 were collected by Richard Randriamam- 2.1 %±0.5% (18 informative sites), and 2.3%±0.3% (54 pionona, Jean C. Randriamanana, Gerard Nalanirina, informative sites), respectively. and Edward Louis on 25 November 2004,26 November 2004, and 26 November 2004, respectively. DAR5.1, Distribution.—L. milanoii is currently known adult male; and DAR5.2, female, are live vouchers from the Daraina region, south of the . L. maintained at Parc Botanique et Zoologique de Tsim- milanoii has been identified with molecular data in An- bazaza; 2 x 2.0 mm biopsies from ear pinna and 1.0 cc drafiamena Classified Forest. L. ankaranensis has also of whole blood; tissues stored at Henry Doorly Zoo's been identified at Andrafiamena Classified Forest. Center for Conservation and Research. A Microchip pit tag was placed subcutaneously between scapulae Comparisons and Remarksmilanoii (0.72 of DAR5.1 and DAR5.2 and recorded as 462C010E43 kg) is approximately the same size as L. dorsalis (0.73 and 46506B0114, respectively. DAR5.1 and DAR5.2 kg), smaller than L. ankaranensis (0.79 kg), but larger were collected by Richard Randriamampionona, Gerard than L. septentrional is (0.6 kg). Although L. milanoii Nalanirina, and John R. Zaonarivelo on 18 October has been identified within the range oi'L. ankaranensis 2005. Complete measurements are available in Ap¬ at Andrafiamena Classified Forest, L. milanoii is the pendix la. only species found at Daraina. Additionally, L. milanoii has not been found at Ankarana or Analamera National Type Locality.—MADAGASCAR, Province de Parks which has only L. ankaranensis. The Loky River Antsiranana, Daraina, Andranotsimaty (approximately is currently the northern barrier for Propithecus tatter- 13°08'52.5"S, 049°41T1"E). sally but L. milanoii has managed to cross this barrier, probably towards the river’s source when there was an Description.—L. milanoii is reddish-brown on intact forest track between Daraina and Andrafiamena back and grayish-white on the venter. The head is (Figure 8). Further studies should be conducted south reddish-brown dorsally, but gray brown on the face, of Andrafiamena Classified Forest and the southwestern forming a mask appearance. A diffuse darker brown limit of the Loky River to determine the distribution of midline stripe in found on the dorsum of the head, con¬ L. milanoii. Additionally, extensive population studies tinuing partially down the back. The anterior portion should be conducted at Andrafiamena Classified For¬ of the thighs is reddish-brown, but the majority of the est to determine the population dynamics of the two limbs are gray in color. The pelage is long and thick, species found there. and missing portions of the pelage, especially on the posterior limbs prominently gray, contrasting from the Etymology^The name milanoii is proposed for reddish-brown surrounding fur. The tail is uniformly the Daraina region and is derived from the Malagasy reddish-brown. language and means, ‘‘to swim”. The Malagasy people refer to the Daraina region as “Daraina milanoa” which Diagnosis.—In the D-loop, 12s rRNA, and means “Daraina swimming” in reference to their swim¬ PAST sequence fragments, L. milanoii differs from its ming in the Andranotsimaty River looking for gold. closest relatives by geographic and genetic distance, L. ankaranensis, L. septentrional is, and L. dorsalis, 26 Special Publications, Museum of Texas Tech University

Vernacular Names.—Daraina or Swimming located in primarily deciduous thicket or thorn scrub Sportive Lemur. (spiny) and in the limited gallery forests. L. leucopus is found in the spiny desert forest parcels of Andohahela Lepilemurpetteri. New Species National Park. Further studies should be conducted north of Onilahy River to determine the northern extent Type Series.-Whole blood for BEZ15 (TK125511/ of L. petteri. Additionally, further studies should be TTU104444), adult female; BEZ18 (TK125563/ conducted in the remaining forest regions around the TTU104496), adult male; and BEZ21 (TK1255 12/ Linta and Menarandra Rivers to determine the distribu¬ TTU 104445), adult female; are stored and curated at tion of both L. petteri and L. leucopus (Figure 8). The Museum of Texas Tech University. Individual measurements, e-voucher photos, and collection data Etymology.—The name petteri is proposed in are available online (see Appendix la) and from The honor of Jean-Jacques Petter for his immense body Museum of Texas Tech University under the respective of work on lemurs, including the sportive lemurs. TTU catalog numbers. BEZ15, BEZ18, and BEZ- Considered a leader in French , Dr. Petter 21 were collected by Richard Randriamampionona and was a winner of the WWF Gold Medal in 1981 for his Edward Louis on 5 March 2001,5 March 2001, and 6 conservation work in Madagascar. March 2001, respectively. According to Jenkins (1987), a specimen, ZD. 1892.111.61, (skull) is held at the Brit¬ Vernacular Names.—Petter’s Sportive Lemur. ish Museum of Natural History from the Ambolisatra Forest (23°03'S, 043°24"E) which is located within the Lepilemur seali. New Species tentative range of L. petteri. Type Series. —Whole blood for JAR2 (TKI25523/ Type Locality.—MADAGASCAR, Province de TTU 1 04456), adult male; JAR3 (TK I 25524/ Toliary, Beza-Mahafaly (approximately 23°39T 1.4"S, TTU 104457), adult female; and JAR8 (TK125525/ 044°37'90.6"E). TTU 104458), adult female; are stored and curated at The Museum of Texas Tech University. Individual Diagnosis.—In the D-loop, 12s rRNA, and PAST measurements, e-voucher photos, and collection data sequence fragments, L. petteri differs from its closest are available online (see Appendix la) and from The relative by geographic and genetic distance, L. leuco- Museum of Texas Tech University under the respec¬ pus, by 3.4%±0.8% (21 informative sites), 1.1%±0.3% tive TTU catalog numbers. JAR2, JAR3, and JAR8 (9 informative sites), and 1.8%±0.2% (46 informative were collected by Richard Randriamampionona, Jean sites), respectively. C. Randriamanana, Gerard Nalanirina, and Edward Louison 19 July 2001, 19 July 2001, and 21 July 2001, Distribution.—L.petteri is currently known from respectively. the Beza-Mahafaly region, south of the Onilahy River and west of the Linta and Menarandra Rivers. Type Locality.—MADAGASCAR, Province de Antsiranana, Anjanaharibe-Sud Special Reserve (ap¬ Description.—L. petteri is larger than L. leucopus proximately 14°47r45.1 "S, 049°27'88.5"E). with a similar gray to grayish-brown body (Table I). The venter is whitish-gray. The face is gray with lighter Description.—L. seali is a large-sized sportive circular patches around the eyes and under the chin. lemur (0.95 kg) with a uniform light chocolate-brown The ears are trimmed in lighter fur with the anterior to reddish-brown color pattern. The venter is lighter or inner lining of the ear dark brownish-gray which brownish-gray and the pelage is extremely long and highlights the ear. There is diffuse brownish-gray thick throughout the body. The face is light brownish- pelage on the anterior aspect of the thigh and along gray and is uniform in color. The hands and feet are a the dorsal midline. lighter grayish-brown. The tail, in contrast to the rest of the body, is brownish-gray throughout its length. Comparisons and Remarks.—L. petteri (0.63 kg) is larger than L. leucopus (0.54 kg). L. petteri is Louis, Jr. et al.— Analysis of the Sportive Lemurs 27

Diagnosis.—In the D-loop, 12s rRNA, and (TK 125536/TTU104469), adult female; are stored and PAST sequence fragments, L. seali differs from its curated at The Museum of Texas Tech University. closest relative by genetic distance, L. muste/inus, by Individual measurements, e-voucher photos, and col¬ 10.1 %± 1.1% (70 informative sites), 4.4%±0,6% (43 lection data are available online (see Appendix la) and informative sites), and I0.7%±0.6% (274 informative from The Museum of Texas Tech University under sites), respectively. In the D-loop. 12s rRNA, and the respective TTU catalog numbers. L0K04.2, PAST sequence fragments, L. seali differs from its LOK04.5, and L0K04.33 were collected by Richard closest relatives by geographic distance, L. wrighti, a Randriamampionona, Jean C. Randriamanana, Gerard newly described species found at Kalambatritra Spe¬ Nalanirina, Rambinintsoa Andriantompohavana, and cial Reserve, by 9.2%±1.1% (79 informative sites), John R. Zaonariveloon 6 July 2004, 6 July 2003, and 4.3%±0.6% (37 informative sites), and I0.1%±0.5% 9 July 2004, respectively. (242 informative sites), respectively. Type Locality.— MADAGASCAR, Province de Distribution.—L. seali is currently known from Antsiranana, Nosy Be, Lokobe National Park (approxi¬ the Anjanaharibe-Sud region, north of the Antain- mately 13°23'27.6"S, 048°18T5.2"E). ambalana River. Description.—L. tymerlachsoni is light brown¬ Comparisons and Remarks.—L. seali (0.95 kg) is ish-gray with the upper half of the back a lighter red¬ approximately the same size as L. mustelimis (0.99 kg) dish-brown. The venter is a light grayish-white. The and L. wrighti (0.95 kg). Although L. seali is located anterior aspects of the thighs and edges of the extremi¬ closer geographically to L. mustelimis, L. seali is taxo- ties also have a light reddish-brown diffuse color. A nomically closer related to the southeastern sportive dark brown to black midline dorsal stripe is present lemur, L. wrighti (Figures 2-8). Additional survey work from the head to the lower half of the back. The face is required to determine the southern range of L. seali is gray with the appearance of a mask. The tail is an and the northern extent of L. mustelimis. Currently, we uniform light reddish-gray to brown. have assigned the sportive lemur from Mananara-Nord (south of the ) to L. seali even Diagnosis.—In the D-loop, 12s rRNA, and PAST though the available molecular data suggests that this sequence fragments, L. tymerlachsoni differs from its population will be described as a separate species in closest relatives by both genetic and geographic dis¬ the future, pending further field studies. Further studies tance, L. milanoii, L. ankaranensis, L. dorsalis, and L. also should be conducted north of Anjanaharibe-Sud scihamalazensis, by 2.5%±0.6% (18 informative sites), Special Reserve to determine the northern extent of L. 3.9%±0.8% (25 informative sites), 4.9%± 1 % (33 infor¬ seali (Figure 8). mative sites), and 4.4%±0.9% (30 informative sites); 2.1%±().5% (18 informative sites), 2.2%±0.5% (20 Etymology>. —The name seali is proposed in honor informative sites), 2.2%±0.5% (18 informative sites), of Dr. Ulysses Seal, an architect of the Conservation and 2.5%±0,5% (20 informative sites); 2.3%±0.3% (54 Breeding Specialist Group SSC/IUCN, and a definitive informative sites), 2.3%±0.3% (55 informative sites), leader, teacher, and motivator of in situ conservation 4.7%±0.4% (110 informative sites), and 4.8%±0.4% throughout the world. (126 informative sites), respectively.

Vernacular Names.—Seal's Sportive Lemur. Distribution.—L. tymerlachsoni is currently known from the Lokobe region and Nosy Be Island. Lepilemur tymerlachsoni, New Species Comparisons and Remarks.—L. tymerlach¬ Type Series.—Whole blood for L0K04.2 soni (0.88 kg) is larger than L. milanoii (0.72 kg), L. (TK 125535/TTU 104468), adult male; L0K04.5 sahamalazensis (0.70 kg), L. dorsalis (0.73 kg), L. (TK125537/TTU104470), adult female; and LOK04.33 ankaranensis, and L. septentrionalis (0.60 kg). Andria- 28 Special Publications, Museum of Texas Tech University holinirina et al. (2006) presented data that distinguished a mask-like appearance. For both sexes, the ears have

L. sahamalcizensis from L. dorsalis (Ambanja/Nosy minimal to short fur and are lighter in color. Be), but the morphological and molecular data clearly separate L. dorsalis (Manongarivo/Antafondro) and L. Diagnosis.—In the D-loop, 12s rRNA, and PAST tymerlachsoni (Nosy Be), and is comparable to differ¬ sequence fragments, L. wrighti differs from its closest ences between the other northern recognized sportive relatives by genetic distance, L. seali and L.fleuretae, lemurs (Figures 2-8). by 9.2%± 1.1% (79 informative sites) and 10.2%±1.2% (70 informative sites); 4.3%±0.6% (37 informative sites) and 3.l%-fc0.5% (27 informative sites); and Etymology.—The name tymerlachsoni is pro¬ posed in honor of the Howard and Rhonda Hawk 10.1%±0.5% (242 informative sites) and 10.8%±0.5% Family who have made generous contributions to the (187 informative sites), respectively. In the D-loop, 12s Madagascar Project, providing infra-structure and field rRNA, and PAST sequence fragments, L. wrighti differs support to the many Malagasy graduate students. from its closest relative geographically, L. leucopus, by genetic distance of 13.9%±1.4% (99 informative sites), 3.1%+().5% (27 informative sites), and 16.4%-H).8% Vernacular Names.—Hawk’s Sportive Lemur. (395 informative sites), respectively.

Lepilemur wrighti, New Species Distribution.—L. wrighti is currently known from Kalambatritra Special Reserve, west of the Mananara Type Series.—Whole blood for KALA4.9 River and north of the . (TK125527/TTU104460), adult female; KALA4.16 (TK125565/TTU104498), adult male; and KALA4.18 Comparisons and Remarks.—L. wrighti (0.95 (TK125526/TTU104459), adult female; are stored kg) is a large sized sportive lemur, approximately and curated at The Museum of Texas Tech University. the same size as L. mustelimts (0.99 kg), but larger Individual measurements, e-voucher photos, and col¬ than L. fleuretae (0.80 kg). With its close taxonomic lection data are available online (see Appendix la) and relationship with the northeastern sportive lemur, L. from The Museum of Texas Tech University under seali, and its distinctive morphological appearance, the respective TTU catalog numbers. KALA4.9, the species is well differentiated from other sportive KALA4.16, and KALA4.18 were collected by Richard lemurs in eastern Madagascar. Further studies need Randriamampionona, Jean C1. Randriamanana, Gerard Nalanirina, Rambinintsoa Andriantompohavana, and to be conducted to determine its distribution (Figure Edward Louis on 12 June 2004, 12 June 2003, and 14 8) and to verify the possible pelage difference between males and females. June 2004, respectively.

Type Locality.—MADAGASCAR, Province de Etymology.—The name wrighti is proposed in Toliary, Kalambatritra Special Reserve, Befarara (ap¬ honor of Dr. Patricia Wright for her long term dedica¬ tion and contributions to conservation in Madagascar proximately 23°25'05.4"S, 0461>26'55.4"E). and tropical environments throughout the world.

Description.—L. wrighti is a large-sized lemur with a diffuse reddish-brown and gray pelage. The Vernacular Names.—Wright’s Sportive Lemur. dorsum is reddish-brown to grayish-brown. The venter is grayish-brown, lighter in color than the dor¬ As with all of the lemur taxa of Madagascar, sum. The head of females is uniformly gray, sharply comprehensive census and taxonomic studies of contrasting with the rest of the body. The males have free-ranging populations in the Genus Lepilemur are a diffuse reddish brown to gray appearance that does necessary for the implementation of management and not contrast dramatically with the body, Thus, based conservation programs. The existing distribution records for all of the Lepilemur species are limited on our limited sample size (n = 5), L. wrighti represents the first instance of sexual dichromatism in the genus (Thalmann and Ganzhorn 2003). Furthermore, the molecular data from previous studies regarding mul¬ Lepilemur. Furthermore, in some individual females, there is a slight color change around the face that forms tiple lemur species reflects specific biases to regions of Louis, Jr. et al.— Analysis of the Sportive Lemurs 29 the mitochondrial DNA; and therefore, data sets based defined regions. Superimposition of the new Lepilemur on independent vouchers or sample sets are difficult to species’ distributions on a geophysical map indicates that correlate (Andriaholinirina et al. 2006; Pastorini et al. the rivers are forming significant barriers, contributing 2003; Wyner et al. 1999; Yoder et al. 2000). Although the to the processes of speeiation. Additionally, an inverse sequence data presented in this paper are not all-inclusive relationship appears to exist between body size and nor equivalent to every lemur mtDNA study or region, number of species with the smaller body sized genera the authors for the first time present sequence data from containing the greater number of species. This effect three regions of the mtDNA in an attempt to submit not appears to be more amplified in the nocturnal lemurs only new data, but also to correlate the data with previous which have smaller territories than the larger bodied studies. By developing an equivalent data set with re¬ diurnal lemurs, with rivers forming the limiting boundar¬ spect to the various regions of the mtDNA sequenced by ies for each species. The eastern Lepilemur species also the various researchers, a comprehensive and cumulative appear to have less dense populations than the species approach could be created that would link independent found in northern and western Madagascar. Correlating but complementary research programs. Microcebus and Avahi sequence data to the distributions of the 22 Lepilemur species, a geophysical map is cre¬ The processes of evolution are an important com¬ ated which indicates a comparative partitioning based ponent of species concepts, science, and investigation. on unique species, with their distributions bounded by The PSC not only encompasses a cladistic perspective, the same river systems or pairs (unpublished data; Louis but also allows the biologist to focus operationally on the et al. 2006). The known distributions of the mid-sized results of evolution to present and to delineate species. A diurnal lemurs, for example, the Genus Eulemur, also cumulative approach implementing morphological, cyto¬ corresponds to the Lepilemur species distributions. Even genetic, and molecular data would be the ideal approach a cursory glance at the geophysical map and the distri¬ when defining species (Mayor et al. 2004; Mittermeier et butions of the 22 sportive lemurs, allows researchers to al. 2006). The fine-tuning of dynamic population studies predict areas where new species likely exist; therefore, will require constant and consistent addition of nuclear more effectively concentrating research efforts (Figure and mitochondrial DNA data from multiple individu¬ 8). als from all geographic ranges to this data set. Results derived from such a data set would be instrumental in The extreme degree of regional endemism, making accurate, precise, and responsible management the continued risk from human encroachment, and decisions by wildlife and conservation entities. the restricted geographic ranges combined with the inadequate knowledge of established distributions of By applying the phylogeographic studies by Pas¬ described lemurs across Madagascar, emphasizes the torini et al. (2003) and Thalmann (2000), geographic need for further investigations in unexplored sites and barriers can be defined that affect multiple taxa, including the re-evaluation of protective measures, conservation both flora and fauna. By establishing and prioritizing ranking, and management of these species. The limited these phylogeographic regions, conservation manage¬ knowledge of geographic limits should be a major prior¬ ment decisions can be implemented that will support ity for conservationists and scientists alike. the preservation of biodiversity within these critically

This manuscript was supported in part by a grant Parc Botanique et Zoologique de Tsimbazaza, U. S. from the Committee for Research and Exploration of Fish & Wildlife, and the Ministere des Eaux et Forets the National Geographic Society (6613.99), of Madagascar. We would like to thank Primate Con¬ Action Grant, and Margot Marsh Foundation Grant. servation, Inc., for their initial support. We acknowl¬ This project would not have been possible without the edge the generosity of Bill and Bemiece Grewcock for support of the staff, guides, and drivers of the Institute their long-term support and commitment, which gave for Conservation of Tropical Environments, Madagas¬ the CCR its direction and identity. Furthermore, we car (ICTE-MrCET), as well as the Association Natio¬ would like to acknowledge that this research would nal pour la Gestion des Aires Protegees (ANGAP), not be possible without the incredible support by the 30 Special Publications, Museum of Texas Tech University

Ahmanson Foundation, the Theodore F. and Claire specialists, Gary D. Shore, Kelly Herrington, Patrick M. Hubbard Family Foundation, and the James Fam¬ Lill, Dana Gilbertson, and Ron Kipple for creating the ily. We would also like to acknowledge the computer web page and documents.

Literature Cited

Amato, G., J. Gatesy, and P. Brazaitis. 1998. PCR assays of Geng, H., D. Bastola, and H. Ali. 2004. Anew approach to clus¬ variable nucleotide sites for identification of conserva¬ tering biological data using message passing. Proceedings tion units: an example from Caiman. Pp. 177-190 in of the 2004 IEEE Computational Systems Bioinformatics Molecular approaches to ecology and evolution (R. Conference 2004:493-494. DeSalle and B. Schierwater, eds.). Birkhauser Verlag, Geng, H., X. Deng, D. Bastola, and H. Ali. 2005. On cluster¬ Basel, Switzerland. ing biological data using unsupervised and semi-super¬ Andriaholinirina, N., J-L. Fausser, C. Roos. C. Rabarivola, vised message passing. Proceedings of the 5th IEEE 1. Ravarimanana, D. Zinner, U. Thalmann, J. U. Gan- Symposium on Bioinformatics and Bioengineering zhom, B. Meier, R. Hilgartner, L. Walter, A. Zaramody, 2005:294-298. C. Langer, T. Hahn, E. Zimmermann, U. Radespiel, M. Gonzales, S., J. E. Maldonado, and J. A. Leonard. 1998. Con¬ Craul, J. Tomiuk, I. Tattersall, and Y. Rumpler. 2006. servation genetics of the endangered pampas deer (Ozoto- Molecular phylogeny and taxonomic revision of the ceros bezoartiens). Molecular Ecology 7:47-57. sportive lemurs (Lepilemur, Primates). BioMed Central Evolutionary 6:1-13. Groves, C. P. 2000. The Genus Cheirogaleus: unrecognized biodiversity in dwarf lemurs. International Journal of Baker, C. S., A. Perry, J. L. Bannister, M. T. Weinrich, R. B. Ab- Primatology 21:943-961. emethy, J. Calambokidis, R. H. Lien, J. U. Lambersen, O. Ramirez, P. Vasquez, J. Clapham, A. Ailing, S..). O’Brien, Groves, C. P. 2001. Primate taxonomy. Smithsonian Institu¬ and S. R. Palumbi. 1993. Abundant mitochondrial DNA tion, Washington, D.C. variation and world-wide population structure in hump¬ Hedges, S. B. 1994. Molecular evidence for the origin of back whales. Proceedings of the National Academy of birds. Proceedings of the National Academy of Sciences Sciences 90:8239-8243. 91:2621-2624.

Bandelt, H-J, P. Forster, and A. Rohl. 1999. Median-joining Huelsenbeck, J. P, and F. Ronquist. 2001. MRBAYES: Bayes¬ networks for inferring intraspecific phylogenies. Mo¬ ian inference of phylogeny. Bioinformatics 17:754-755. lecular Biology and Evolution 16:37-48. Ishak, B.. S. Warter, B. Butrillaux, and Y. Rumpler. 1992. Barrowclough, G. F„ and N. R. Flesness. 1996. Species, subspe¬ Chromosomal rearrangements and speciation of sport¬ cies. and races: the problem of units of management in ive lemurs (Lepilemur species). Folia Primatologica conservation. Pp. 247-254 in Wild in captivity 58:121-130. (G. G. Kleinian, M. Allen, and H. Harris, eds.). University of Chicago Press, Chicago. Illinois. IUCN/SSC Criteria Review Working Group. 1999. IUCN Red List Criteria review provisional report: draft of Cracraft. J. 1983. Species concepts and speciation analysis. the proposed changes and recommendations. Species Pp. 159-187 in Current ornithology, Vol. 1 (R. F. Johnston, 31-32:43-57. ed.). Plenum Press, New York. Jenkins, P. D. 1987. Catalogue of primates in the British Davis, J. 1., and K. C. Nixon. 1992. Populations, genetic Museum (Natural History) and elsewhere in the British variation, and the delimitation of phylogenetic species. Isles. Part IV: suborder Strepsirrhii, including the sub¬ Systematic Biology 41:421 -435. fossil Madagascan lemurs and Family Tarsiidae. British Felsenstein, J. 2005. PHYL1P (Phylogeny Inference Package) Museum (Natural History), London, England. version 3.6. Distributed by author. Department of Ge¬ Jernvall, J., and P. C. Wright. 1998. Diversity components nome Sciences, University of Washington, Seattle. of impending primate . Proceedings of the Forster, P., A. Torroni, C. Renfrew, and A. Rohl. 2001. Phy¬ National Academy Sciences 95:11279-11283. logenetic star contraction applied to Asian and Papuan Kappeler, P. M., R. M. Rasoloarison, L. Razafimanantosoa, L. mtDNA evolution. Molecular Biology and Evolution Walter, and C. Roos. 2005. Morphology, behaviour and 18:1864-1881. molecular evolution of giant mouse lemurs {Mina spp.) Gray, 1870. with description of a new species. Primate Report 71:3-26. Louis, Jr. et al.— Analysis of the Sportive Lemurs 31

Kimura, M. 1980. A simple method for estimating evolutionary Pastorini, J., R. D. Martin, P. Ehresmann, E. Zimmermann, and rates of base substitutions through comparative studies M. R. J. Forstner 2001. Molecular phylogeny of the of nucleotide sequences. Journal of Molecular Evolution lemur family (primates) based on mito¬ 16:111-120. chondrial DNA sequences. Molecular Phylogenetics and Evolution 19:45-56. Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: Molecular Evolutionary Genetics Analysis, Version 2.1. Pennsylva¬ Pastorini, J., Thalmann, U., and R. D. Martin. 2003. A molecu¬ nia State University, University Park Press, Baltimore, lar approach to comparative phylogeography of extant Maryland. Malagasy lemurs. Proceedings of the National Academy of Sciences 100:5879-5884. Longmire, J. L., G. F. Gee, C. L. Hardekoff, and G. A. Mark. 1992. Establishing paternity in whooping cranes (Grus Petter, J.-J., and A. Petter-Rousseaux. 1960. Remarques sur americana) by DNA analysis. The Auk 109:522-529. la systematique du genre Lepilemur. Mammalia 24:76- 86. Louis, E. E. Jr., M. S. Coles, R. Andriantompohavana, J. A. Sommer, S. E. Engberg, J. R. Zaonarivelo, M. 1. Mayor, Petter, J.-J., R. Albignac, and Y. Rumpler. 1977. Faune de and R. A. Brenneman. 2006. Revision of the mouse le¬ Madagascar 44: Mammiferes Lemuriens (Primates, murs (Microcebus) of eastern Madagascar. International Prosimiens). ORSTOM/CNRS, Paris, France. Journal of Primatology 27:347-389. Pope, T. R. 1996. Socioecology, population fragmentation, Maddison, W. P, and D. R. Maddison. 1992. MacClade: and patterns of genetic loss in endangered primates. Pp. analysis of phylogeny and character evolution. Sinauer 119-159 in Conservation genetics, case histories from Associates, Sunderland, Massachusetts. nature (J. C. Avise and J. L. Hamrick, eds.), Chapman and Hall, New York. Martin, R. D. 2000. Origins, diversity, and relationships of lemurs. International Journal of Primatology 21:1021 - Posada, D., and K.A. Crandall. 1998. Modeltest: testing the 1049. model of DNA substitution. Bioinformatics 14:817- 818. Mayor, M. 1„ J. A. Sommer, M. L. Houck, J. R. Zaonarivelo, P. C. Wright, C. Ingram, S. R. Engel, and E. E. Louis, Jr. Rasoloarison, R. M., S. M. Goodman, and J. U. Ganzhorn. 2000. 2004. Specific status of Propithecus spp. International Taxonomic revision of mouse lemurs (Microcebus) in the Journal of Primatology 25:875-900. western portions of Madagascar. International Journal of Primatology 21:963-1019. Mayr, E. 1942. Systematics and the origin of species from the viewpoint of a zoologist. Columbia University Press, Ravaoarimanana, B. 2001. Genetic comparison of wild popula¬ New York. tions of Lepilemur septentrionalis and Lepilemur dorsalis using RAPD markers. Primates 42:221-231. Mittermeier, R. A., I. Tattersall, W. R. Konstant, D. M. Mey¬ ers, and R. B. Mast. 1994. Sportive lemurs. Pp. 83-94 Ravaoarimanana, B., R. Tiedemann, D. Montagnon, and Y. in Lemurs of Madagascar. Conservation International, Rumpler. 2004. Molecular and cytogenetic evidence for Washington, D. C. cryptic speciation within a rare endemic Malagasy lemur, the northern sportive lemur (Lepilemur septentrional is). Mittermeier, R. A., W. R. Konstant, F. Hawkins, E. E. Louis, Molecular Phylogenetics and Evolution 31:440-448. O, Langrand, J. Ratsimbazafy, R. Rasoloarison, J. U. Ganshom, S. Rajaobelina, l. Tattersall, and D. M. Mey¬ Ronquist, F., and J. P. Huelsenbeck. 2003. MRBAYES 3: ers. 2006. Sportive lemurs. Pp. 183-208 in Lemurs of Bayesian phylogenetic inference under mixed models. Madagascar, 2nd Edition. Conservation International Bioinformatics 19:1572-1574. Tropical Field Guide Series, Washington, D. C. Rumpler, Y., and R. Albignac. 1975. Intraspecific chromosome Montagnon, D., 1. B. Ravaoarimanana, and Y. Rumpler. 2001. variability in a lemur from north of Madagascar: Lepil¬ Taxonomic relationships and sampling effects among emur septentrionalis, species nova. American Journal of Lepilemuridae and Lemuridae using a partial cytochrome Physical Anthropology 42:425-429. b gene. Cytogenetics Research Academy of Science, Rumpler, Y.. B. Ravaoarimanana, M. Hauwy, and S. Waiter. Paris, Sciences de la vie/Life Sciences 324:647-656. 2001. Cytogenetic arguments in favor of a taxonomic Myers, N. 2000. Biodiversity hotspots for conservation priori¬ revision of Lepilemur septentrionalis. Folia Primato- ties. Nature 403:853-858. logica 72:308-315.

Nylander, J. A. A. 2004. MrModeltest v2. Program distributed Sambrook, J., E. F. Fritch, and T. Maniatus. 1989. Molecular by the author. Evolutionary Biology Centre, Uppsala cloning: a laboratory manual, 2nd edition. Cold Spring University. Harbor Press, New York.

Pastorini, J., M. R. J. Forstner, and R. D. Martin. 2000. Rela¬ Saitou, N. and M. Nei. 1987. The neighbour-joining method: tionships among brown lemurs (Eulemurfulvus) based on a new method for constructing phylogenetic trees. Mo¬ mitochondrial DNA sequences. Molecular Phylogenetics lecular Biology and Evolution 4:406-425. and Evolution 16:418-429. 32 Special Publications, Museum of Texas Tech University

Schneider, S., D. Roessli, and L. Excofier. 2000. Arlequin: a Thalmann, U., and T. Geissmann. 2005. New species of woolly software for population genetics data analysis. Version lemur Avahi (Primates: ) in Bemaraha 2.000. Genetics and Biometry Laboratory, Department (Central Western Madagascar). American Journal of of Anthropology, University of Geneva, Switzerland. Primatology 67:371 -376.

Schwarz, E. 1931. A revision of the genera and species of Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and Madagascar Lemuridae. Proceedings of the Zoological D. G. Higgins. 1997. The ClustalX-Windows interface: Society of London 1931:399-426. flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research Smith, R. J., and W. L. Jungers. 1997. Body mass in comparative 25:4876-4882. primatology. Journal of Human Evolution 32:523-559. Wheeler, Q. D., and N. 1. Platmck. 2000. The phylogenetic Swofford, D. L. 2001. PAUP*. Phylogenetic Analysis Using species concept {sensu Wheeler and Platnick). Pp. 55-69 Parsimony (*and Other Methods). Version 4.0b5. Sinauer in Species concepts and phylogenetic theory: a debate (Q. Associates, Sunderland, Massachusetts. D. Wheeler and R. Meier, eds.). Columbia University Tamura, K., and M. Nei. 1993. Estimation of the number of Press, New York. nucleotide substitutions in the control region of mito¬ Wyner, Y. M., G. Amato, and R. DeSalle. 1999. Captive breed¬ chondrial DNA in humans and chimpanzee. Molecular ing, reintroduction, and the conservation genetics of black Biology and Evolution 10:512-526. and white ruffed lemurs, Varecia variegata variegata. Tattersall, I. 1982. The primates of Madagascar. Columbia Molecular Ecology 8:S 107-S115. University Press, New' York. Yang, Z., and S. Kumar. 1996. Approximate methods for esti¬ Telenius, H.,N. P. Carter, C. E. Bebb, M. Nordenskjob, A. J. Pon¬ mating the pattern of nucleotide substitution rates among der, and A. Tunnacliffe. 1992. Degenerate oligonucle¬ sites. Molecular Biology and Evolution 13 :650-659. otide-primed PCR: general amplification of target DNA Yoder, A. D., R. M. Rasoloarison, S. M. Goodman, J. A. Irwin, by a single degenerate primer. Genomics 13:718-725. S. Atsalis, M. J. Ravosa, and J. U. Ganzhorn. 2000. Thalmann, U. 2000. Lemur diversity and distribution in western Remarkable species diversity of Malagasy mouse lemurs Madagascar - inferences and predictions using a cladistic (Primates, Microcebus). Proceedings of the National approach. Pp. 191-202 in Endemismeetdiversite a Mada¬ Academy of Sciences 97:11325-11330. gascar, Memoires de la Societe de Biogeographie (W. R. Zhang, D.-X., and G. M. Hewitt. 1996. Nuclear integrations: Loureneo and S. M. Goodman, eds.). Paris, France. challenges for mitochondrial DNA markers. Trends in Thalmann, U., and J. U. Ganzhorn. 2003. Lepilemur, sportive Ecology and Evolution 11:247-251. lemur. Pp. 1336-1340 in The natural history of Mada¬ Zimmermann, E., S. Cepok, M. Rakotoarison, V. Zietemann, gascar (S. M. Goodman and J. P. Benstead, eds.). The and U. Radespiel. 1998. Sympatric mouse lemurs in University of Chicago Press, Chicago, Illinois. northwest Madagascar: a new rufous mouse lemur spe¬ cies {Microcebus rave/obensis). Folia Primatologica 69:106-114.

Authors 'Addresses:

Edward E. Loljis, Jr. Shannon E. Engberg

Center for Conservation and Research Center for Conservation and Research Henry Doorly Zoo Henry Doorly Zoo 3701 S, 10,h St. 3701 S. l(Yh St. Omaha, NE 68107, USA Omaha, NE 68107, USA E-mail: edlo@omahazoo. com E-mail: genetics@omahazoo. com E-mail: (Lab - United States): [email protected] Email: (Field - Madagascar): [email protected] Louis, Jr. et al.— Analysis of the Sportive Lemurs 33

Runhua Lei Gisele Randria

Center for Conservation and Research Departement de Paleontologie el d’Anthropologie Bi- Henry Doorly Zoo ologique. 3701 S, 10th St. University of Antananarivo Omaha, NE 68107, USA BP 906 E-mail: [email protected] Antananarivo 101, Madagascar E-mail: [email protected]

Huimin Geng

Prosper Department of Pathology and Microbiology University of Nebraska Medical Center Parc Botanique et Zoologique de Tsimbazaza Omaha, NE 68198, USA BP 4096 E-mail: hgeng@mail. imomaha. edu Antananarivo 101, Madagascar E-mail: prota. madagascar@wanadoo. mg

Julie A. Sommer

Borome Ramaromilanto University of Nebraska, School of Biological Sciences 315 Man ter Hall Parc Botanique et Zoologique de Tsimbazaza Lincoln, NE 68588, USA BP 4096 E-mail: juliesommer@yahoo. com Antananarivo 101, Madagascar E-mails: [email protected]: prota.madagciscar@

Richard Randriamampionona wanadoo.mg

Center for Conservation and Research Gilbert Rakotoarisoa Heniy Doorly Zoo 370I S. 10th St. Parc Botanique et Zoologique de Tsimbazaza Omaha, NE 68107, USA BP 4096 E-mail: sma/lvaovao@omahazoo. com Antananarivo 101, Madagascar E-mails: prota. madagascar@wanadoo. mg; gilbertra-

Jean C. Randriamanana kotoarisoa@yahoo. com

Center for Conservation and Research Alejandro Rooney Henry Doorly Zoo 3701 S. HD St. National Center for Agricultural Utilization Research Omaha, NE 68107, USA U. S. Department of Agriculture E-mail: smallvaovao@omahazoo. com 1815 N. University/ St. Peoria, IL 61604 USA

John R. Zaonarivelo E-mail: ROONEY@ncaur. usda.gov

University> of Antananarivo Rick A. Brenneman BP 906 Antananarivo 101, Madagascar Center for Conservation and Research E-mail: [email protected] Henry Doorly Zoo 3701 S. 10th St.

Rambinintsoa Andriantompohavana Omaha, NE 68107, USA E-mail: [email protected]

University of Antananarivo BP 906 Antananarivo 101, Madagascar E-mail: [email protected] 34 Special Publications, Museum of Texas Tech University

Appendix I

The following Appendices to this publication are available online at the indicated website addresses.

a. Lepilemur Field Data (Individual data file for each Lepilemur, including morphometries, photos, sequence accessions, global position system, microchip data, gender, and location):

http://10.10.10.3/ccr/genetics/lemur/index.asp?page=ccr/genetics/lemur/Megaladapidae.htm

b. Map Figure 1 (Maps of historical distributions):

http://www.omahazoo.com/ccr/genetics/papers/appendixl.pdf

c. Primer Table 1 (Summary of designed primers for D-loop, 12s rRN A, and PAST fragments):

http://www.omahazoo.com/ccr/genetics/papers/appendixprimertablel.pdf

d. Pairwise Data Table II (Complete pairwise matrices):

http://www.omahazoo.com/ccr/genetics/papers/appendixtablell.pdf

e. Pairwise Aggregate Analysis Figure 2 (Complete PAA for Summary of the D-loop, 12s rRN A, and PAST fragments):

Figure 2A, D-loop PAA - http://10.10.10.3/ccr/genetics/papers/d-loop.pdf

Figure 2B, 12s rRN A PAA - http://10.10.10.3/ccr/genetics/papers/rRNA.pdf

Figure 2C, PAST PAA - http://10.10.10.3/ccr/genetics/papers/pastorini.pdf

f. Haplotype Summary List Figure 3 (Summary List of the Lepilemur Haplotypes for D-loop, 12s rRNA, and PAST fragments):

http://www.omahazoo.com/ccr/genetics/papers/appendixIH.pdf

g. Sequence Summary Table III (Summary of the D-loop, 12s rRNA, and PAST fragments):

http://www.omahazoo.com/ccr/genetics/papers/appendixtablelIl.pdf

h. Bayesian D-loop Figure 4 (Bayesian analysis of the D-loop for 211 Lepilemur)-.

http://www.omahazoo.com/ccr/genetics/papers/appendixfigure4.pdf

i. Message Passing Cluster Figure 5 (Message Passing Cluster analysis of D-loop for 211 Lepilemur):

http://www.omahazoo.com/ccr/genetics/papers/appendixfigure5.pdf Louis, Jr. et al.— Analysis of the Sportive Lemurs

a- >n SO n oo SO so sD so so m rn m m m a. Os Os OS OS OS OS o SO X X X X X so o a r- n- c- n r- r-- so C- t"~ > >- >- >- > < < < < < < $

a- SO in fN in m H cm n >n in >n 2 £ <: $ «? % $ $ $ < $ 5 «S 5 a s 5

a- m 00 Os O rx| rn a- 00 00 0 O in so a 00 Os O ___ SO so SO SO SO so fN rsj ^j- fN so so SO so so 3 SO SO a- S? £ 5 § <

3 3 3 3 3 cd § CD CD § § 3 3 3 3 3 CD cu Q 3 3 3 I 5 2 3 3 3 _0> I -I o ~3 i I 3 -2 a> c 3 3 % 3 3 3 3 3 <3 3 3 3 1 3 3 3 3 a, o 3 3 3 3 3 3 3 3 cx \s 3 g a g 3 ct is 3 3 3 3 3 g g c c 3 3 u tofl J5 s i 3 i 1 S g 1 fc -Si ~Si "Si -Si -Si ■2 "Si 3 a> & s* s- 'S’ & S' S’ *§• & 1 '§’ ’I’ x U Q a <3 <1 -3 <1 a a S’ S’ '1 '1 NH <1 <1 a a a a a a a Q X u CL. •S •5 •S •2 yi a. d> ■5 •5 ■S •S •2 3 I s LO £5 § 1 3 § 3 S 3 CD CD CD <3 3 3 CD i 53 CD CD 53 § < £ 3 3 3 3 3 CD ?3 3 3 3 3 3 3 2 2 2 i 5 3 3 | anensis 3 2 3 3 -2 -2 -2 4 ^2 3 3 3 i | ~a | 1 -*: •a ■2 a 1 3 3 § 3 3 3 3 3 3 S 3 3 3 3 3 3 3 3 3 3 cx J V 3 s 3 3 3 g fv g g 3 3 3 3 3 3 3 £ s s 3 3 3 ! I 4> 3 s 1 1 s .2P'S j 1 ~Si ! CD S-

03 03 a ^ a ^ ^ Lh !■, jSS o u <3 o tj u j] g .a a ~ aa-s -O a xi axia X) ax>*■* c a 5 1 -O ! -SO 3JSs.2S.s|*i.3§§ -2 5 .2 S J3 03 a' 13 a Sc’aa'Sa'Sn^eos o C < c a < < < <<<<^<|<|<|

X> a 2

H H H

ra to 3 fN Os —1

o o o sO SO O' O' Cl, o O SO n n O r- >n in a a a a a a a a a a a a a a $ < Q Q Q a Q a a Q Q Q Q Q a

—3- 3-O) t-~ r- O' O' H CO a a a a a a a a a a a a a a £ $ $ < < a Q Q Q Q Q a Q Q Q a Q Q Q

O' sC r- O', r-i 3- 3- 3- m n rt (N n >n >n mi >n »n in Z 3- 3"

fij 5 | 3 3 3 <3 3 3 _a) 44 .8 4< I 'o jg Jg t '53 3 a> 'I S* 1 S- S' 'I ¥ S' ST s* S' S S' S' S' U Q <1

3 <3 £ 3 443 s 3 I 3 R R R g R 4&fR JS

S' s- S' 1 S' S' 'S' 1 s S' RJ> ‘S' S' S' O Q -4 <1 -4 -4 -4 -4 -4 ^4 *4 -d

3 3 3 3 3 3 3 3 3 3 3 3 3 3 d 3 E E Z E £ E £ £ E E S' 3 3 3 S' 3 S' ;d s at 3 s 3 O 0 13

-C £3 z iZ iZ (—1 H

00

_ o _ CN m N in NO r- OO On ON dr in NO r- On r-- 00 oo r- r~ 00 oo 00 00 00 oo oc OO ON o o o o nO r-- r- in in m in in m m in m in m df in m in in >n ”d- N" p, ON ON O' ON ON O' ON ON ON O' ON ON ON ON ON ON ON ON ON Q rs r-i n 0-1 (N CN (N n in >n >n >n in >n >n in >n >n >n m >n in m >n m in o o o o o o o cx O' a a CX a a a o a a O' Q Q Q a Q Q Q Q Q Q Q Q Q Q Q Q c a Q Q

m (N m N- m NO r- oo ON o NO r- oc O' o o ■—i r^H m n m m m in >n >n in m in in m m m m a a a a a a a a a a Cx cx O' a a a O' a o

PAST' Q Q Q Q Q Q Q Q Q Q Q Q a a a Q Q Ci Q

o NO r~' oc oi m m ro df n- N- N^f df N- N ’^t o ON ON ON ON ON ON ON ON ON O) n 03 03 03 (N 03 03 03 < in m in >n m in in m m CX O) cy O' O' O o O o Cx CX o O O o O o o o o Q Q Q Q Q Q Q Q Q Q Q Q Q a Q a Q Q Q

•2 ■S •2 ~2 "a 2 a a o o o o a a a a a o cy cu do 2 2 .be .be %> .fao s & 1 & 1 5 a cd K K a Z S .|t 3 .2 J5 .2 o is '«! & Si, u 3 'I s- S’ s* S' & S' Ssi ¥ & s* U Q -3 xl <1

X 5 2 2 z <3 o a a w n. ■§ ■§ "§ -§ ■as c. 2 P p p p p Cy o a. & & 0) _ a £X n c/o .2 3 cd K a g .£ ou _2 _2 JS ,2 jg .SP 'S S, I- (U s- S' & 'I ¥ S’ S' & 'I S’ S’ S’ O Q xi X3 <1 --3 •-1 -J >, "cd cd B cd cd . ’C > > '5 -S o _cd c 5 cd 2 cd x“ fc? ST e2 &T* ^ >-, -2 cd X) X) X3 -J8 M <3 ja a jd « k Id ,St 13 § -1—’ Cd ^p-* rrt M—< (Tj M—< —-J H—< • - . sa S ^ cd cd I I cd 43 1^1 t3 C na 4> JB 43 g c cd rf\ cd rn cd c/n cd

ui y! H H H [—1 i- H H H

NO 00 ON 03 00 o NO 03 rn df; ro in >n (N N- N" mi O' 6JQ fe *T) mi in mi >n W W < < < < < < N N- ^ aa >- >n .< < i—3 -1 J OS oS cd £ X OS Z OS < < < cd 3 < < < < < < % I I < U Z c lx lx tx (X -1 -1 _l £ £ 2? 2 Q Q 38

Catalog TK Number* Location Original Species Current Species 12s PASTb D loopb Number Designation Designation rRNAb

DAR4.18 TK125564 Daraina Lepilemur ankaranensis Lepilemur milanoii DQ529331 DQ529621 DQ529479 (Andranotsimaty) cn on a M3 CN cN Q ''t a >n CN Q ON Q a Q Tf '—‘ ON < ✓—n csvrs'—' "U SoTO^3"O30 M g.i o n-j2^ n iN ON NO m a Q Q CN cn Q aJ < A 2 3 St § £ g 5? or Cl, 3 <3 cd o 2 a cN Q ON NO n- a Q Q a I cn Q c4 A r- < a 3 3 a c a CN ON n- NO o m n rf a a >n O' NO CN a Q -2^ i-j j§ CN Q (2 o A < s 5 3 v. S Special Publications,MuseumofTexasTechUniversity 1 L1 c3 575 e d c o oo Q a O' >n in cn ■d" a ON NO CN Q in CN o a -§ N o E d d cd NO a ON 00 Q *n CN NO CN ON cn Q a Cx >n Q I ’3 Q < (X tT Tf S' g § g 3 cu 3 a 3 a 2 d cd d O E cd >. § VI 1 '3 _d Q Si mi < 3 g g g cd & 3 Vh 3 . S' T3 Id Q j- d co E d S3 J 1 -3 "E _d Q (2 CN m < 5 g <4> £ S 3 p cd 3 d3 O E od d § CO C**> SO On Qs r- < —i »n ON CN NO CN $ $ J5 1 •2 i —4 H O c4 < £ g 00 d > o o d cd S' . CN On >n ON o Q a n ON ex CN r- Q m a in CN ON. ■*? Q n- ~3 jg ~§ <2 TO Z H mi |n < *2 g g £ d cd TO _d3 .2 -a ? §2 o2 d d g 5d 2c3 CN ON CN a n O' a CN Q a Q Tf tj- 1 ”s •2 z H mi NO < s2 £ 3 ON CN ON cn Q a in >n a n- Q Q «n CN n- a ~§ ON <1 .g CN (- * Z H mi r < £ £ S' 3 TO "S' d CM E cd cd 2 CN ON Tt- a >n ON in Q On Q a CN Tf tT a Q n Q a a cn ON N" Tf a a in ~C '1 JO dX3Z CN mi < N < 3 g g 3 £ 3 cd MZg C3 gCj2a, £ cdcox> CN NO a ON in ON a m On a CN r- a >n CN Q a ON Tf Tf NO >n -3

o (N NO NO NO X NO NO NO NO nD ■'tf- ^r ^t rr ON ON O' Q, ON O'. ON ON ON O NO

ri '*$■ NO ON o o o o o o o o X NO NO NO NO NO NO NO NO NO NO X NO NO NO NO ON ON ON O' ON ON ON On ON ON On On O' On ON H a a a a a a a a Q Q Q Q Q Q Q Q Q < $ a Q Q Q Q Q Q a

m _ _ < c« Z nf ?n od a a a a a a a a a lx & a a a a a a a a a Q Q Q Q Q Q Q Q < < Q Q Q a a a Q a

-s: NJ 5S -3 2 is § 3 3 K a g ^3 & £* s 1 9$ 1 4 4 cti g g H c c 5 s £ go5 JS Z 'I S' & S' & S' ‘I S' S' S' S' u U C -**} ►-j <1

X 3 z W .52 a- •2 •2 ~£s -3 “?s 2 1 “$s &< ~3 "3 Jo fn a a s a < £ 3 3 O 4 4 4 I 4 ex 'a "^s 1 I 00 . g g g g s c K g S s OX) S j§ S, ex eg ex, I 'S' 'S' S- S' S' 'S' S' S' & S' nT 5r §- §- O Q ►>q *-q *-q <1 ►■X <3 <1 '-J n-J

t/3 o o O o X X X r~l X o M M M 'm 'm (U 1) o

x X X x X x (—1 h-1 H (—1 H H H

O' o rn r) cn on NO on fN r-4 rn rn rn cn oc on oo '=^' ■^r (N so (3 X n on X X o X o O o o o o o o O O o O o X 2 Q£ 2 cd g u xi x: X X CQ PQ ffl CQ < <: < < c3 3 o o o o o o c o o z z u z _q -q _1 nJ -1 < < X 2 X X 2 Special Publications, Museum of Texas Tech University

,__ 00 ON o 04 rO in NO r-~ oo ON o 04 ro ref nO NO O' o- O- O' O' i-' r^ C4C oo OO ON On On On O ref ref ref Nit ^t ■^t ref ref ref ref ref ref 0\ o ON On On ON ON ON ON ON ON O' On O' ON On O o< 04 04 04 04 04 04 04 04 04 Ol 04 04 04 04 04 04 04 2 in in in in in in >n >n >n in >n m, in in m in m —( a a a a a a a a a a a a a a a a a G Q Q Q Q o a Q D a Q a Q a Q Q G Q

o oi rO ref >n NO oo 'On o 04 CO ref in nC oi on rn rO CO CO ro m NO NO NO nC sD NO NC nC NO NO NO NO NO NO NO NO NO On O' ON On ON ON ON ON On ON ON ON ON ON o o o 04 oj 04 O) 04 04 o) 04 04 04 Ol 04 04 04 04 04 04 m m m m m >n ■n in in m in m m in GO >n >n in a a a a a a a a a a a a a a a a £ Q Q G a Q a Q a Q Q Q a a Q Q Q Q

© Ol m ref >n NO O' oo QN o 04 CO ref m NO n oi Ol Ol 04 04 04 04 04 on "3- ref ref ref ref ref ref ro m rO rO m m ro rO rO on m m CO CO CO CO m _c ON ON. On On ON O'. O' ON ON ON ON ON ON ON ON o ON Ol oi 04 04 Ol 04 04 04 04 ol Ol oi 04 04 04 04 04 < in m >n in in in m m m m «n in in m >n m

rRh Q Q a a Q Q n o Q a Q Q a Q Q Q Q

n NJ N4 £ g 5 g g 1 1 I O p rdsi rdsi rdsi Ss s. s. g.

p $s p § rcocki o o o' p p p 4^ g g N4 'Sj O g ~S fa & 1 I’ p1 ¥ ¥ S' ¥ ¥ ¥ S' ¥ ¥ 1* U Q oi <1 ■-j —i i-J

g I | 1 1 1 S, s s. 03 I 1 % §- S' s. g. (D § P 5 a p o P o p 7- NJ CJ o g gj s g g P CJ R o> _ 1 5l> S s $4 P "1 "1 P "1^4 ^4 H4 ~s a £ * 5^, *. 5^ co .2 r r 2s a S S 55 s S3 5; g g s 5 s 544 R Sr SC 2l S s s s R R IS4 J5 g4 g4 • S §> J5 -S J3 .SP-53 G

K3 KS CO c C 03 ca C3 cd 03 re3 cd res a t—4 t- M res ■ res M -JS _2 '5b '5b OX) JS — res .2 03 03 J3 o o O o o o .2 13

1. n m m m ir> m m in oi 04 o-J oi r<\ oi 04 oi 2

2 2 5 3 2 3 3 3 3 H H f—1 H H h f—1 H f—1

be oi rn o NO oo >n NO r- oo ON oo O 04 o) rO O rn 04 rn NO NO NO NO NO o- o o' ai c4 Ol 04 a a 2 G Q Q Q Q Q Q Q B S X! < < < < < < X X a3 G 2 2 2 2 2 2 2 2 2 'Zl < U 2 2 X X < < < < < < < AND93 Andohahela Lepilemur leucopus DQ529347 DQ529637 DQ529495 AND94 Andohahela Lepilemur leucopus DQ529348 DQ529638 DQ529496 Louis, Jr. et al.— Analysis of the Sportive Lemurs 41

O' 0 — 0) co 4 'A, 43 00 t" O 0 LA, vO 4 la 43 vO VO 'O vo 43 VO VO sO O' 4 O, O' O' 'A, la. CD >/-, la 'Al 'Al 'A, 'Al 'A, CO >A O', CO m O' O' O', O' O' O' O' O' O', O' D- O' O' O' O' O' 04 04 04 04 04 04 04

A) IA) IA3 IAS *A3 'A 'Ai *A3 'A 'Ai 'A, *A 'Al LA LA, LA LA LA, a a a a a a a a a a 4 4 4 4 4 a 4 4 >« Q Q Q Q Q Q Q Q Q 0 < < < < < Q < < <

O' 0 c^i rA 4 *A3 vo 00 4 04 CO 4 'A 0 0 oc O' O O' O' O' O' O' O' 04 4 4 4 4 4 4 4 4 4 4 4 n 04 04 O' Ov O', O' O' O' O' O' O' O' 4 4 4 4 4 O' 4 Ol r'l C! r-i n A4 Ai >Ai IA3 *Al IA LA 4 4 4 4 4 4 4 4 Z a a a a a a a a a a Uh U_ to U_ a u. U. Uh X a G Q Q Q Q Q Q Q G < < < < < Q < < <

3 3 3 4 4 §■ I I £ P P p 'O 4 JS P P P i> c 4 4 4 4 4 Cu O m -p, 3 K H 5 Z -§ <4 o 4 4 4 u $ £ £ 1* £ £ £ £ £ 'I £ 5T iT U Q **4 ►4 -4 -4 4 4 4 4 4 4 4 4 4 4 4

z 3 z 3 w 3 3 -S 0. -§ 4 ~§ 0. 4 £ £ £ §: p p P p <4 < P I

3

*S1 6bs .P bo •- s\ Cx, 4 4 'C

ja >> ,KS ,KS ^3 «3 4" CSJ 4x >;g >;g n 5 q i/30Jc/3

— co 01 — vC —

Z U Z Z Z H f—1 4 H 4

vO 4 oc O' 0 04 CO r- 04 *A -0 —; 4 4 4 04 Oj 04 ro CO CO OO jj vO •A >A 'A >A >A 'Ai >A >A o| 04 04 4 LA oc 04 _, O', c _c LAS 0 O O O 0 O O O O 0| 04 O O ~a £ O N N N N N N N N N N N N N N X z z z 3 3 N < < < < < < < < < UJ X W X X X 4 < < 0 z < 'X X X X X X X X X X X X X X X X CZ 42 SpecialPublications,MuseumofTexasTechUniversity

Catalog TK Number3 Location Original Species Current Species 12s PASTb D loopb Number Designation Designation rRNAb

RAN02.24 Ranomafana Lepilemur microdon Lepilcmnr microdon AY585736 DQ529678 DQ529536 (Vohiparara) a C! Q On in |p a CN NO r- ON m oo in m r- p o in r^- >n *§ <3 < ~P ^5 ,03 CN C4 -4 ¥ o IN in p P B s i P 03 p p 5 0 cd o S 1 p s: H p a p p r—s 3 ic £ cd 2 2 a CN ON n o- a CN NO On Q m Q p O o a (N ON $■*. >n m N" p ,cd I p) .P & p3 .P 0^ W CN 2 s 1 P g K p O o 03 03 g P g e G P »33 S cd C5 C-I i—i oo a on a o o m CN m m, oi On o a o 00 p in r~~ CN ON p in Tf* ¥ -Si P4 ,G ,P oj 73 ¥ & < rb P 0 a p 1 g p cd 5 s P P P 03 o £ G p K c a. cd 2 2 s Os SO rn OS < >- 00 (N SO Os a\ > o < in o (N in tj* < 1 ►-3 4(-l ■P .Nj pj ed N 'S' Pu CQ £—i I O K 1 V a s p K cd C O £ 03 p p P G S a S£ 3 l 1 grt 2 s SO OS r*"> Os 00 >H oo 04 On ON C (N > >n •§ Uh t— in M < ~p < 1 ’o pj m H O G r-i (p H CN in >n n a On oo m 04 NO o Q ON oo p >n a $£* cd cd s & p o C cd c £ g p p a p i£ “s' t 1 ex ON n •p Nj -Si "§ .cd .c > O G CN NO & P3 ,p g p s p cd cd p a cd c O G a g cd 2 G, 2 NO ON k r- o m 04 t}“ •n OO NO o < lu r- -Si < _G -Si 3 NO § H < p] •2 & cd G G g p cd £ p p p p o < >- NO ON Tf > •n oo ri >n (N < Uu r- < 4G -Si ncp. 5 ~p ? CN NO p3 s H (—1 >n m IP Cj P G cd £ 0) G 03 P g P 4T ON o in >- r- O') NO < NO -d" < > in NO o oo CN N o m >- in -Si < ~p ~P -G <1 3 1 y\ H 04 S4 CN in in pj PQ N H m ,—i ¥ G < 1- s <43 Nj S ^3 cd £ C 53 p P !< P 13 a oo a o) ON in 04 Q >n a o o p >n CN NO r~- ON to oo «n 04 o p a 3 _cd pj -p ~p •5 ON G aq H .to N ¥ < <3 P g cd £ D G cd S p a p p ON CN O' >n 04 ON a a >n a o) NO — >n r- a 04 O' m 00 p p •n —i -4 3 -G 1 -Si *—5 p-i .jo < < 'd" oo L NJ p Ci3 <43 cd g P cd £ 1» G 03 43 5 U o a in CN On >n m On NO cn Q a 04 p in F- CN On -3 Xi 1 -P ■—i s CN p) .-o < ¥ < SJ p £3 s !3 cd £ D G g 52 P G P P Ti¬ a •n CN ON in ro NO a p m G P 1 p P p p p

JAM4.27 Anjahamena Lepilemur aeeclis Lepilemur aeeclis DQ529387 DQ529677 DQ529535 Appendix II (cont.) Louis, Jr.etal.—AnalysisoftheSportiveLemurs u o Q z u a C/) ’G ri d £ 00 t—' cd cd fc C/5 S .1 o. o o> g co Z o cx o Z 1 X) < o g G aj O') < Z ^3 Q o '1 c a CN ,U O' t-+- o CO) Q O' Q a 1 P XI E< O & s | S -cO g 4p < Z •—> N rn 02 H -4 Z 'I a Q 02 O' N O' cco a a o\ 02 Q O' o R P 1 X 14 << S -5 0 g cd u v) 0 44 < •—> N m m -4 H Z ‘1* a O' o Q n O'. CO) CO) Q a co, (N ot o ho Q O' oi O' >0) dt O' oo P X S P S S' < O CZS & £ pO O on Id Z g i. < < 2 dt oo oo < o- HO 5 hO O' dt o Sh t- 0 55 cd G C oj § P 3 | X "m O 00 44 < Z m H < -4 ‘s- Z dt oo < U, dt O' o~ CN £ HO O $ HD Oh O- o dt G 0 (U cd !_ ig X 5 3 pH 1 £4 CQ U4 f— CN CQ ~4 i *-4 Uh & o~ dt O' o < dt CN O' $ HO o 5 3J £ 04 G O g P P Sp X 1 S’ 44 u- y o Uh T3 ■C -g < <2 ¥ O' ,3 < Uh dT r- dl- HO 5 $ HO O' 3 t- dt o O' cd cz c cd a Pp g 5 HP 4 < -a ’C Uh z f—1 Uh Z ¥ IN Uh dT O' dt- < | < O' $ r- HO dt G G 2 G G 3 p S 3 S' dt Uh < z CN dt t— Z Uh gU 1 gj a (N <1 O O O .4) Q a ‘S’ Q CN O' co) hO O' o a cd G G cd C 5 p 3 e o p 3 ¥ O O dt 'P _o JP N < 44 -4 ~3 a OI as rn -4 Q a Q lO) O' dt CN HO a Q 2 N 0 3 4 R g 5j >5 X S' c X> Id -2 < CO £ 0 G G G G 1 S -4 1 js -P -4 u- df r- dT CN o .3 < Z < < Z1 0 § 0 £ 3 3 3 P R R I pH X R Xi m ¥ d}- -4 Uh CN O < o~~ df r| .3 < 5 O' r- HO dt S 0 £ 0 3 3 R P O R S' cq _G l -3 U- dt O'] o C" dt m < 5 O' r- HO dt § 0 £ 0 4 3 p R 3 3 S' S £ ~3 CQ 2 “■4 "1* O') <3 Ux r^~ .3 dt s < dt 5c 5 O' o~ HO dt 0 0 £ G 3 R P O R P CQ ,0 s *■4 ’3p dt r~- 02 < Uh dt O CO) £ 5 G c 0 C E 4) R p 3 5 3 o X CQ dt -4 1 "4 Uh dt r- dt n o .0 < HO 5 o 2 iO) oo HO < Z HO OH t" dt s 0 s 0 3 3 i: R R S' pX) CQ 1 "§ h4 ^4 dt dt < Uh t' r-i O 5 'E 5 oo — < 0 0 g 4) 3 R g S R S’ pX) H Z 1 2 P4 -4 -4 < dt uo dt

re n so rt >n sC Cl rt rt rt rt ci ci CN in in in ri cl ri rt in m m m m rt rt rt rt in m >n rt rt it O' O' O' O' O'. O' O' O' O' ON O' OS On O' O' O' O' C'} ci ci ci CN SO SC sO ci ci ri SC sO sO n| n ^ 1 m m >n in r-~ C' r~ r- >n >n >n C' r~ r~ a a a a o a >- >- > a o a a >- >- a a a Q Q Q a < < < Q Q a Q < < < Q Q Q o a

rt m sO C~- oo O' o .—i rt n SO so 00 oc OC oo oc oo Os O'. CN CN m On o- o sC sO sO sc sC so sC SO SO SO SO sO sO X x O' O' O' Os O' O' CN CN m O' On Os O' O' O' ci ci ri ci Cl C-l m ON CN OO 00 oo in n in >n >n n in in a a a a > z a a a a a o a a a >- a Q Q < < < Q a Q Q <

m in so rt >n SO m ro n rt so o O O' O' O' Os O' O' ci o C ri m CN ro rr, r#i m m rt rt rt rt ri rn ci r-i CN O' O' O' O' O' O' O'. O' O' ^t rt rt O' rt cl CN c-l m t*- r- Cl Cl Cl Cl Cl Cl r~ < 1C, n >n •n >n in >n in rt rt rt in rt rt rt ifl Z Uh a o a a a a a u, lu Uh (N a cy o a U- LLh a a Q Q Q Q < Q Q Q Q < < < Q Q Q Q a < <

M t5 '53 ft o O

* 3 z w PH Is PH

a> _ cn.2 13 2 .S §) 3 .Sp-a ft ft ft, >- a> 1 'S ¥ ftc O & & S' c\T O Q <3 --4 ft ft -4 ft ft -4 ft -4

N .2 ■a G S s _o O © O o o G c3 "c1 'c1 'g1 3 £ 3 f— N

4^ H f— - H

IN OS rn oo O' o c- oc SO sC —< >n Cl in in SO sq re cn mi rt w) s ■—i o X SO Cl rf rt rt rt rt rt rt rt rt Cl < < < < Cl Cl z 2 S N N N N Q Q Q Q Q a Q Q a Q s DC z z < < < < < < < < < < cn cn < u z § (S) H (— h- E—1 (- i— H H t—1 h- H N Louis, Jr. et al.— Analysis of the Sportive Lemurs 45

oo o

00 os o —s -- CN o CN >0 rj rd n rn cn o sO d" sC oo Os O o sO so SO sC sC SO X X x x sO sO SO SO sO so sO so sO sO SO sO sO CN r\ rs)

33 ,R i § “I ■ ■I ■53 13 ~33 13 13 ~a 3 33 3 3 3 3 3 a a 1 "33 ~3 “53 <3 -a ~C3 z a Z, z. <1 ~C3 ai

IZ 52 ’S a rZ a Q .§ z .a a 1 ■§ 1 1 1 fZ | 3 ! | | ■f | | W CA Z) a a Oh 0) s a a a a a a 3 3 s >3 52 52 o u a a aj a a cO Oh o z, Z, ■ 4 r:; Z 'C, a> — 1 a rZ ' f a^ < 5 a a C a £ C/2^ .dc !v a £ a a a a a a a a a a a R a g a a d ^ i 1 .£ too 3 ~3 -Si -3 -3 ! too ‘55 1 -3 i -3 1 1 -§ 1 'C o> % | &■ 1 §* '1- ‘S’ O Q Z z z % '§- '1- ‘1 i z z z z Z z 1 z z z Z Z Z z

cd cd -o 2 cd e3 cd o £ £ E 0) oo 0) CQ CQ CQ es cd cd d d cd a> (U 0) 0) a> , Z E X X X X X 1 c too too too X x X X x X X 1—1 2 2 £3 d d s £ n £ £ £ E x cd cd

r-~ (N r-

vO 3 O' O' O' O' 3 3 O' o' o o 3 3 3 vO 3 O' 3 3- 3 3 3 3 DO & oouo — m o >- a a a c/ Q Q a Q 5c «3 < < < 5 < <5 $ <5 5c

(N 3 3 3 3 3 O' sO vo sC 'C O' O' O'* O' H < Ol Olyn CO Z a a c/ a £ 2 5 < < % 5c 5c 5 £ 5 pH Uh 5c & Q Q o Q 5= 5c 5

(T, (3 3 CN rj CO r- r- Tf */") oo O' O' 0 i/to >r, 'r, 0 CO ro CO CO CO CO O' 0 CO ■*}- 3" r rn CO co --•4- 0 r j CM rl n r- CN 0-1 o\ Ol n 0-1 O' O' O' On ^r ■'3- '^I- -1- 3- 3- C'l 3- 3 O'] Ol Vto t" r-- 00 00 r- 00 0 p. O) n r, V) CTi >0, uo 'r, xt- ^1- Ti¬ Ti¬ 3- 3- 3 !-G to. Ul. Cg a a a O CG u_ to. to- to_ > z >- < < < < < < < < 5 < < a Q n Q < < < <

O > .>5 3 ~5 5 ^2 .1 p Q Cs-j XT-, 5 ~p p to ’a to M 4^ pto ■£ to) 5 5 s S 3 tos 1 ’p s3 Q tos % .p' Co p P *3 3 ‘CJ s> 3 G 'ZJ Co >5 Co 5 2 to. .50 •5fj § 3 S &, _o V Cl -§ s g CO to) to) t- i? s 3 i 0 .2 to) to 2) to c G S3 .to S p P ~p p H bl) 1 S3 is ’S, ’S. Si p to .to Z fc Tfi rs 3 "a P 3 3 * Sr 1 1 S' k5 o 1 cC § § 1 t 1 1 u U Q to) -to to) ^4 <1

* S) S z -C - Cu s "to 0, s= toSo

.fee ■8 ,$p "3 g R S! g 5 5 .to 1 i to .£ (5fl to ~§ p' tos tos to! .£P 5S p C 3 p §■ & 1 & 1 I I O Q *4 <1 >*4 »-4 3 I

"O T3 T3 33 03 3 3 3 3 3 CO CO CO CO co T3 JD ‘C ^ W C g 3 a 3 5 i* « M 'a 3 E O £ £ _3 £ £ G 2 £ £ toto o 3 3 g o o o C o o ’c1 c o -G ,C G S-l G G M 3 G c -G X -G o cj G 3 C 3 G G C _tocd k" '.u03 3 3 3 G 3 3 < < < < < 2 o * Qi < c* PQ OP < 3 &5 e* qP

U H h-

o Ol oo Ol 01 O' cO tf co cn m 'O »/o oi 00 O' CC 3 01 'O co OO 3 co co co fcfl c CO cc. 3 < O 0 o sO o n O' o o CO CO CO c* Z (Z Z Z z z Z z z 2 £ c* cZ Di £Z o < 3 3 < < < < < < £ 5 u z JARS )—5 1—> Z 2 ai 2 < Louis, Jr. et al.— Analysis of the Sportive Lemurs 47

o (N oo O' o O' O' o 3- 3- 'cf "d- Oh 3- 3- o oo ^1- o »/~> CM <: $ 5c <2

(N ^j- m m VO VO »0 VO) vr> vr> n 3 >“ >« >< < < < < <3

VO oo O' o vO i-3 (N r-- t"- r, 3 > Uh ? < < < < < <

b5 a I ?| 3 bo bo oa a 'ft* S bo bo bo bo a a a a GO -Z3 a a a a c3 C C S 5 i H

£ K a 3 z $ UJ 3o C. bo bo o cu o a < 'S' 1 S S bo bo cnj a a S -32 I -22 bS bs 'a bs I 1 3 CD O Q I I | I 5 5

kS (30 (30 S c s c o o o o c e c c cn c as < 02

O'