la 1 W TEXAS TECH UNIVERSITY Natural Science Research Laboratory Special Publications Museum of Texas Tech University Number 51 20 June 2007 Molecular Phylogeny and Taxonomic Revision of the Woolly Lemurs, Genus Avahi (Primates: Lemuriformes) J/KM# Hit r i yi'iW h»il 1 .■ r- S UiJirniri'u fji ^ ,1m ;-r i rJr.'UJJ t)ffw *- C ^vjin rjM&aunt NWTpnd (i .ItwJh' I'jvriii^r Uli'twlnr -trnfri Ffftxt r ■^1 ri illSj 1XCVI Ji'Ajili'l I Rambinintsoa Andriantompohavana, etal. Front cover: Revised distribution of the woolly lemurs of Madagascar. The revisions of the sportive lemurs (Louis et al. 2006b) and this publication demonstrate the partitioning of unique Avahi and Lepilemur species by rivers which act as barriers. Each region is defined by a distinct woolly and sportive lemur. Figure created by Kelly Herrington and Edward E. Louis, Jr. Special Publications Museum of Texas Tech University Number 51 Molecular Phylogeny and Taxonomic Revision of the Woolly Lemurs, Genus Avahi (Primates: Lemuriformes) Rambinintsoa Andriantompohavana, Runhua Lei, JohnR. Zaonarivelo, Shannon E. Engberg, Gerard Nalanirina, Susie M. McGuire, GaryD. Shore, Justin Andrianasolo, Kelly Herrington, Rick A. Brenneman, and Edward E. Louis, Jr. University of Antananarivo and Henry Doorly Zoo Layout and Design: Jacqueline Chavez Cover Design: Kelly Herrington and Edward E. Louis, Jr. Copyright 2007, 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: 20 June 2007 Library of Congress Cataloging-in-Publication Data Special Publications of the Museum of Texas Tech University, Number 51 Series Editor: Robert J. Baker Molecular Phylogeny and Taxonomic Revision of the Woolly Lemurs, Genus Avahi (Primates: Lemuriformes) Rambinintsoa Andriantompohavana, Runhua Lei, John R Zaonarivelo, Shannon E. Engberg, Gerard Nalanirina, Susie M. McGuire, Gary D. Shore, Justin Andrianasolo, Kelly Herrington, Rick A Brenneman, and Edward E. Louis, Jr ISSN 0169-0237 ISBN 978-1-929330-12-6 Museum of Texas Tech University Lubbock, TX 79409-3191 USA (806)742-2442 Molecular Phylogeny and Taxonomic Revision of the Woolly Lemurs, Genus Avahi (Primates: Lemuriformes) RambinintsoaAndriantompohavana, RunhuaLei, John R. Zaonarivelo, Shannon E. Engberg, Gerard Nalanirina, Susie M. McGuire, Gary D. Shore, Justin Andrianasolo, Kelly Herrington, Rick A. Brenneman, and Edward E. Louis, Jr. Abstract Approximately 3,000 base pairs of mitochondrial DNA sequence data (control region or D-loop, along with a fragment including a partial segment of cytochrome oxidase subunit 111 gene to Leucine-tRNA gene), morphological and phenotypic characters were used to investigate the phylogenetic relationships among the recognized woolly lemurs (genus Avahi) and to other lemur genera of Madagascar. The molecular data support the previously recognized Avahi species, but it has also revealed additional unique biodiversity. Here, we present further taxonomic revisions of the genus Avahi , by elevating the two recently described subspecies, A. m. meridionalis and A. m. ramanantsoavana , to species level, along with a description of one new species. We also conducted the first phylogenetic analysis of all recognized species of woolly lemurs, all repre¬ sentatives of the family Indriidae, and present the relationships between the recognized species of the genus Avahi and this newly described species. These results underscore the urgency to initiate further detailed studies in previously unstudied sites throughout Madagascar in order to better define lemur species. Key words: Avahi , D-loop, Madagascar, prosimian, systematic, woolly lemurs Introduction Due to its unique species biodiversity and the con¬ tinued pressure from human encroachment in the form of habitat destruction and fauna and flora utilization, Madagascar has been placed at the top of conservation priority lists, or hotspots (Myers 2000). Distributed throughout the island, the prosimians of Madagascar are particularly susceptible to extinction risks due to their relatively small, fragmented geographic ranges, and the potential loss through edge effects and constant habitat loss (Jernvall and Wright 1998; Reed 2004; Ezard and Travis 2006). Since 2000, multiple molecu¬ lar genetic and morphological studies w ithin the genera Avahi , Cheirogaleus, Lepilemur, Microcebus , and Mina have led to a significant increase in the number of recognized species (Zimmermann et al. 1998; Groves 2000; Rasoloarison et al. 2000; Kappeler et al. 2005; Thalmann and Geissmann 2005; Andriaholinirina et al. 2006; Andriantompohavana et al. 2006; Louis et al. 2006a, 2006b; Zaramody et al. 2006). With the addition of newly described species, the once broad ranges of the previously recognized nocturnal species have dramatically decreased and concurrently made the risk of extinction of both recognized and newly described lemurs much greater (a smaller distribution and a diminished number of represented subpopula¬ tions). Currently, all lemurs are protected under the Convention of International Trade of Endangered Spe¬ cies (CITES) and are designated by the 1UCN/SSC Red List Categories from critically endangered to threatened (IUCN 2004), Recent revisions underscore the need to resolve the taxonomy and phylogeny of lemurs so that a scientifically rational approach to their conservation and management can be developed and implemented. Furthermore, the numerous taxonomic revisions of lemurs have led to subsequent modifications of histori¬ cal distributions and population estimates that require a persistent re-evaluation of conservation protection status (Martin 2000; Louis et al. 2006b). 1 2 Special Publications, Museum of Texas Tech University The woolly lemurs (genus Avahi), a group of strictly nocturnal primates that are regarded as pheno- typically indistinct with varying degrees of brown, red, white, and gray pelage, are a prime example of species whose taxonomic/evolutionary diversity until now has been largely unrecognized and underrepresented. Tra¬ ditionally, two species of Avahi have been recognized, one from eastern Madagascar, Avahi laniger , and one from western Madagascar, Avahi occidentalis (Fig. 1). Initially, both species were considered subspecies of Avahi laniger , but morphological, molecular, and cytogenetic differences between the eastern woolly lemur, Avahi laniger , and the western woolly lemur, Avahi occidentalis , have conclusively established spe¬ cies level variance (Rumpler et al. 1990; Thalmann and Geissmann 2000; Zaramody et al. 2006; Figs. 2-4). The potential for additional species within the genus Avahi was predictable, given the discrete and often fragmented distributions, the scattered or spotty occurrence within these fragments, and the widespread area that the genus Avahi is found especially on the eastern coast of Madagascar ( Martin 2000; Thalmann and Geissmann 2000). A comprehensive study to define these populations is critical above and beyond the threat of habitat loss because the highly foli- vorous dietaiy requirements of the woolly lemur can bring about a selective liability that can potentially limit habitat suitability. Consequently, Thalmann and Geissmann (2000, 2005) based on morphological and vocalization data described two additional woolly lemurs from western Madagascar, A. unicolor and A. cleesei , from the Ampasindava region and Tsingy de Bemaraha, respectively (Figs, 1, 5-7). Furthermore, Zaramody et al. (2006) described two new species from the southeastern coast, A. peyrierasi (south of the Mangoro/Onive Rivers to Ranomafana National Park) and A. meridionalis (Manombo Special Reserve to Andohahela National Park; Fig. 1). Avahi meridi¬ onalis was further subdivided into two subspecies, A m. meridionalis and A. m. ramanantsoavana , from Sainte Luce/Andohahela National Park and Manombo Special Reserve, respectively (Fig. 1). Here, we present further taxonomic revisions of the genus Avahi , along with a description of one new species. We also conducted the first phylogenetic analysis of all recognized spe¬ cies of woolly lemurs, all representatives of the family Indriidae, and present the relationships between the recognized species of the genus Avahi and this newly described species. As previously discussed in Andriantompohavana et al. (2006), Louis et al. (2006a, 2006b), and Thalmann and Geissmann (2005), the utilization of whole vouch¬ ers as the designated holotvpe for a new species is not a prerequisite, but that opportunistic collections can later supplement morphological, vocalization, and/or molecular data in combination with curated blood and/or tissue samples. The woolly lemurs are a prime candidate for this methodology because the highly foli- vorous dietary requirements of this group of lemurs cur¬ rently precludes any attempts to curate Hive vouchers” (Thalmann and Geissmann 2005). Total genomic DNA for the three paratype specimens are currently curated at the Museum of Texas Tech University' (TK125757; TK125758; TK 125759). Additionally, an electronic database that includes all Avahi field data and photo¬ graphs, including data for the paratype specimens, is curated at the Museum of Texas Tech University. The database is stored in the Type Specimen Collection in multiple media formats. This collection of field data and photographs, as well as additional tables and fig¬ ures, also are available online at the website of Omaha’s Henry Doorly Zoo. See Appendix I for a directory of appropriate website addresses. (text continued on page 10) Andriantompohavana et al.— Woolly Lemurs of Madagascar 3 Bcmarivo Mahajamha Mariarano^ Betsibolm— Mangoky Mahavavy Nord- Antafondro- Sarnbirano- (MOO kin Macvarano Sofia Mahavavy Slid Maningoza Manambaho Mknambolo Tsingy de Bemaraha Tsiribihina Morondava Fiheruniina Onilahy I radii A ntainambalaria Mananara-Nord Maniri|'ory Onibe Mantadia Maroon izaha Maitgoro Fandriana Namorona Ranomafana Faraony Lima Andohahcla (Manangotry) Maitdrari: A Viihi pcyritrasi P Avahi nr , ramanQntsoavana (P Avahi m. meridionals P Avahi faniger M a no m bo Avahi ittt,caifir Avahi f/rrjn" Avahi oaidvrita fin (P Ftnii l'ijLut of site denotes wesi ccwlki Avahi species, all ftumeriy Avahi ttoritfettialix O' lmii color ol" site denotes cast coast Avahi species, iiLIl formerly Avahi laniger Matiampatrana Figure 1. Sample distribution map of the woolly lemurs of Madagascar. 4 Special Publications, Museum of Texas Tech University Figure 2. Avahi occidentalism western woolly lemur, at Mariarano Classified Forest. Photo by Edward E. Louis, Jr. Andriantompohavana et al.— Woolly Lemurs of Madagascar 5 Figure 3. Avahi occidentalism western woolly lemur, at Mariarano Classified Forest. Photo by Edward E. Louis, Jr. 6 Special Publications, Museum of Texas Tech University Figure 4. Avahi laniger, western woolly lemur, at Mananara-Nord National Park (Ivontaka-Sud). Photo by Edward E. Louis, Jr. Andriantompohavana et al.— Woolly Lemurs of Madagascar 7 Figure 5. Avahi unicolor , Sambirano woolly lemur, at Antofondro Classified Forest (Maromiandra). Photo by Edward E. Louis, Jr. $ Special Publications, Museum of Texas Tech University Figure 6. Avahi unicolor , Sambirano woolly lemur, at Antofondro Classified Forest (Maromiandra). Photo by Rambinintsoa Andriantompohavana. Andriantompohavana et al.— Woolly Lemurs of Madagascar 9 Figure 7. Avahi cleesei, Cleese’s woolly lemur, at Tsingy de Bemaraha National Park. Reprinted with permission of the author and journal (Thalmann and Geissmann 2006). Photo by Urs Thalmann. 10 Special Publications, Museum of Texas Tech University Methods Sampling .—All lemurs investigated in this study were wild-caught (sampling localities are depicted in Fig. 1) and were immobilized with a C0 2 projection rifle or blowgun with 10 mg/kg of Telazol (Fort Dodge Animal Health; Overland Park, Kansas). Four 2.0 mm biopsies and 1.0 cc per kilogram of whole blood were collected from each sedated animal and immediately stored in a room temperature storage buffer (Longmire et al. 1992). The location of the immobilized lemur was recorded using a global positioning system (Ap¬ pendix la). Genomic DNA was extracted from a 2.0 mm ear punch using a phenol-chloroform extraction (Sambrook et al. 1989; Appendix IF). Beginning in 2000, while the animal was sedated, we placed a HomeAgain microchip (Schering-Plough Veterinary Corp.; Kenilworth, New Jersey) subcutaneously be¬ tween the scapulae of each lemur (Appendix II). This procedure was used to field catalog each animal with a unique recognition code to provide for the capabil¬ ity to positively re-identify all captured individuals during any future immobilizations. Measurements were taken on the sedated animals as follows, weight (+/- 0.01 kilograms); head crown ((total length from tip of the nose (not the soft tissue) to the occipital crown)+/-0.1 cm); body length ((total length of body from the occipital crown of the head to the base of tail) +/-0.1 cm); tail length ((total length from base of tail to the end of the last caudal vertebra ) +/-0.1 cm); forelimb measurements (thumb (total length of the thumb (proximal, middle, and distal phalanges) from the distal tip to proximal point of the last bone of the thumb (claw not included) +/-0.1 cm), longest digit (total length of the phalange only (proximal, middle, and distal phalanx, claw and metacarpal not included) +/-0.1 cm), and hand (total length of hand (phalanges and metacarpals included to the carpal joint) +/-0.1 cm)); radius/ulna ((carpal joint (styloid process of the radius) to the olecarnon tip of the ulna) +/-0.1 cm); humerus ((greater tubercle of humerus to the end of the lateral condyle of the humerus) +/-0.1 cm); hindlimb measurements ((toe (total length of the toe (proximal, middle, and distal phalanges not including claw) from tip to insertion point of the last bone of the hind toe (1st digit) +/-0.1 cm), longest digit (total length of the phalange only (claw and metatarsal not included) +/-0.1 cm), foot (total length of hand (phalanges and metacarpals included to the tarsal joint, not including claw) +/-0.1 cm), tibia ((calcaneal tuber to the proxi¬ mal tibial tuberosity) +/-0.1 cm), and femur ((greater trochanter of the femur to the distal point of the lateral condyle of the femur) +/-0.1 cm)); testes ((width and length of the right and left testes in males) +/-0.1 mm); and upper and lower canines ((length of the upper and lower right canines from the tip to the gum line) +/-0.1 mm), For presentation purposes, we present average measurements and standard deviations of the weight, head crown, body length, tail length, forelimb, and hindlimb in this publication following the guidelines of Smith and Jungers (1997; Table 1A-B ). All other measurements, e-voucher photographs, and data are available at http://www.omahazoo.com/ccr/index. asp?page=/ccr/genetics/genhome.htm (Appendix la). All traits were tested for significance among species using Analysis of Variance (ANOVA) performed using an online statistical service (Kirkman 1996). Species means were plotted in histogram form with the appro¬ priate 95% confidence intervals (Appendix Id). Data Collection .—To correlate our data with previously published molecular studies, we analyzed the following regions of the mitochondrial DNA (mtDNA): the displacement loop or control region (D-loop; Baker et al. 1993; Wyner et al. 1999) and a fragment of the cytochrome oxidase subunit TIT gene (COIII), NADH-dehydrogenase subunits 3, 4L, and 4 (ND3, ND4L, and ND4), as well as the tRNA Gly , tRNA Arg , tRNA His , tRNA Ser , and partial tRNA Leu genes (subsequently referred to as the PAST fragment; Pastorini et al, 2000, 2003). Using 50 nanograms of genomic DNA, the D-loop (562-563 base pairs (bp)) and the PAST (2380 bp) fragments were amplified using the following conditions: 94° C for 30 sec, 47° C for 45 sec, 72° C for 45 sec for 34 cycles. Since potential nuclear insertions or mitochondrial pseu¬ dogenes within the nuclear genome can be amplified inadvertently, we chose to minimize this likelihood by amplifying both mitochondrial DNA regions as intersecting or overlapping segments (Zhang and Hewitt 1996). Consequently, the PAST fragment was generated from six amplified segments. Additionally, to further eliminate amplification of nuclear inser¬ tions, a technique that is species independent and both rapid and efficient derived from the degenerate oligonucleotide-primed PCR method (DOP-PCR; Andriantompohavana et al.— Woolly Lemurs of Madagascar 11 Telenius et al. 1992) was used to generate the PCR products. Adapting this LL-DOP-PCR (long products from low quantity), the sequence data were generated for the D-loop fragments and PAST sequence gener¬ ated from overlapping segments were confirmed. The samples were electrophoresed on a 1.2% agarose gel to verify the PCR product and purified using QIAquick PCR purification kit (Qiagen; Valencia, California). Using the BigDye terminator cycle sequencing ready reaction kit by Applied Biosystems the sequence was analyzed by capillary electrophoresis with an Applied Biosystems Prizm 3100 genetic analyzer (Applied Biosystems. Inc.; Foster City, California). A suite of internal sequencing primers from Pastorini et al. (2000, 2001) were used to generate the PAST fragment. Additionally, PCR and sequencing prim¬ ers specific for genus Avahi were designed for the D-Loop fragment, and PCR and sequencing primers specific for ND4 region were utilized for the PAST fragments (Arevalo et al. 1994; Appendix lb). The sequence fragments were aligned to generate a con¬ sensus sequence using Sequencher (Gene Corp; Ann Arbor, Michigan), and the consensus sequences were aligned using ClustalX (Thompson et al, 1997). All aligned sequences are available from the first author upon request. All sequences have been deposited in GenBank and the sequence data and information are available from the referenced accession numbers (Appendix II). Phylogenetic Analysis. —Maximum-parsimony analyses (MP) were performed for the phylogenetic study of the D-loop, PAST, and combined (D-Loop and PAST fragments) sequence data with PAUP Ver¬ sion 4.0b 10 software (Swofford 2001). Heuristic searches were completed using the random addition sequence (1000 replicates) with the tree bisection- reconnection branch swapping routine. Gaps were considered as a fifth character in MP analyses, but were treated as missing data in the NJ analyses. Boot¬ strap analyses were accomplished with 1000, 3000, and 4000 pseudoreplicates with the D-loop, PAST, and combined data sets, respectively. Only nodes with greater than 50% support were reported. The maximum likelihood (ML) analyses were performed using the program PA LIP Version 4.0b 10 software (SwofTord 2001). Due to the large number of taxa and characters and the resulting computational intensity, we pruned the combined sequence dataset by choos¬ ing taxa representing distinct haplotypes supported in an initial neighbor-joining (NJ; Saitou and Nei 1987) analysis. Bayesians inference analyses were conducted using MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The model of evolution was selected by using MrModeltest 2.2, a modified version of Modeltest 3.6 (Posada and Cran¬ dall 1998; Nylander 2004). A Markov Chain Monte Carlo (MCMC) run with four simultaneous chains and 1,000,000 generations was performed. Every hundredth generation, the tree with the best likelihood score was saved, resulting in 4000 trees. The 4000 trees were condensed in a majority rule consensus tree using PAUP Version 4.0b 10 software (Swofford 2001). Branch supports were assigned as posterior probabilities on the consensus tree. The pattern of sequence evolution was estimated by conducting a minimum spanning network generated with the pro¬ gram NETWORK Version 4.11 (Gonzales et al. 1998; Bandelt et al. 1999; Forster et al. 2001) and Arlequin, Version 2.0 (Schneider et al. 2000). In addition to character-based phylogenetic analysis of DNA sequences, PAUP software (Swof¬ ford 2001) was also used to calculate uncorrected pairwise distances for D-Loop, and PAST combined fragments (‘p’)- As described in Davis and Nixon (1992), Wyner et al. (1999), Mayor et al. (2004), Andriantompohavana et al. (2006), and Louis et al. (2006a, 2006b) we utilized MacClade 3.01 (Maddison and Maddison 1992) and MEGA Version 2.0 (Kumar et al. 1993) in a diagnostic search to designate evo¬ lutionary significant units (ESU) using population aggregate analysis (PAA) of the D-loop (562-563 bp) and PAST (2380 bp) sequence data for Avahi. With the sequential addition of each individual without an a priori species designation, a PAA distinguishes attributes or apomorphic characters according to the smallest definable unit (Davis and Nixon 1992; Mayor et al. 2004; Andriantompohavana et al. 2006; Louis et al. 2006a, 2006b). 12 Special Publications, Museum of Texas Tech University Results Following ANOVA among the species, sub¬ species and two additional types, only the tail length was found to be nonsignificant. The body length was slightly significant (P<0.05), the head crown, humerus and femur lengths were moderately signifi¬ cant (PO.Ol), while all other measurements were strongly to highly significant (P<0.001). Means and standard deviations are reported in Tables lAand IB; histograms defining the 95% confidence intervals for all measurements are located in Appendix Id. Mitochondrial DNA sequence data were com¬ pleted for two fragments, D-loop and PAST fragment (approximately 2380 bp) for 98 individuals, repre¬ senting all six recognized species of woolly lemurs from a total of twelve sites (Fig. 1; Appendix II). All new mtDNA sequences generated for this study were deposited in GenBank and can be acquired through the accession number (Appendix la; Appendix II). The sequence alignments for the data sets are avail¬ able from the first author upon request. The PAST fragment consists of the 3' end of the COIII gene (30 bp), the complete NADH-dehydrogenase subunits ND3 (348 bp), ND4L (297 bp), and ND4 (1378 bp), along with the tRNAgenes, glycine (73 bp), arginine (73 bp), histidine (70 bp), serine (65 bp), and the 5’ portion of leucine (47 bp). The polyadenylation of COIII and ND4 genes, insertion of base pairs between ingroup/outgroup comparisons, and other alignment characteristics between lemurs and Homo are consistent with Pastorini et al. (2000). Based on the phylogenetic inferences of the NJ, MP, and ML analyses of three sequence alignments (D-loop. PAST, and D-Loop-PAST fragment com¬ bined), two major Avahi subgroups are represented, differentiating the six recognized woolly lemur spe¬ cies (Figs. 8-14). The first subgroup corresponds to the western woolly lemurs, A occidentalism A. cleesei , and A. unicolor (Fig. 8). The second sub¬ group includes the eastern woolly lemurs, A. me¬ ridional is meridional is, A. m. ramanantsoavana, A. peyrierasi , and A, laniger (Fig. 8). Two haplotype groups or types of A. peyrierasi were represented within the eastern woolly lemurs (Figs. 8-14). In addition, another group of woolly lemurs from the Fandriana region formed a distinct clade, A. species nova #1, from the other recognized species (Fig. 8). There is high bootstrap support for the MP and NJ analysis with respect to the topology of the genera and species as every major node between the species clusters has a greater than 50% bootstrap support (Figs. 8, 11, and 13). The results from the popula¬ tion aggregate analysis of the D-loop and PAST se¬ quence data are presented in Tables 2A-B and 3 A-B, respectively. Multiple diagnostic characters define each Avahi species, along with two types of Avahi laniger (only the results from the PAST fragment PAA designates attributes specific for each region), two types of Avahi peyrierasi , and the one newly described species, A. species nova#l (Figs. 15-17; Tables 2 and 3 ). A review of the morphometric data for the six recognized species of woolly lemurs are summarized in Table 1 and are presented in Appendix Id, along with the two types of A, peyrierasi , and the newly described species (Appendix la; detailed morphological measurements of the individual animals are available at http://www.omahazoo. com/ccr/index.asp?page=/cci7genetics/genhome. htm) . The uncorrected 'p ? distance and the absolute genetic differences are presented in Table 4A-B. The highest average uncorrected pairwise distances and absolute genetic differences for the D-Loop sequence alignment were between the western and eastern woolly lemurs, ranging from 12.28 to 8.84 and 65 to 47 (A. cleesei to A. peyrierasi type #2 and A. oc- cidentalis to A. peyrierasi #3), respectively. The highest average uncorrected pairwise distances and absolute genetic differences for the PAST sequence alignment were between the western and eastern woolly lemurs, ranging from 11.72 to 10.20 and 251 to 220 (A. occidental is to A. m. meridional is and A. occidentalis to A. peyrierasi #3), respectively. The highest and lowest average uncorrected pairwise distances and absolute genetic differences for the D-Loop sequence alignment between the eastern woolly lemurs range from 5.99 to 3.43 and 43 to 19 (A. m. meridionalis to A. laniger and A. species (text continued on page 35) Andriantompohavana et al.— Woolly Lemurs of Madagascar 13 nj > 0 C to & ■n * ^ X cd > < to fc: to- Hw, te “V Ol Si Vj F-* 0 o ' -H w ■M O JO < Q Q -2 w 5* v3 St X £ - Ci to tj N, ^ S 5.: c to P 5 t) 'J 2 § ■§•§ v aj U ^ '*-*+ AJ k o *-. ■** ^ 5/ c <3 £■ K ■S rt t) r* ^ -0 ■§^5 r- ■4 f 0 vc frt V’i r vC a Cv ail r~ ■rt f^j ■—i so O' oc O'. 3 ■w «0 rt /■% o Tl d o ««* o d o d d ri n ri r^ ri d d rn ri s ?-• M rt rt- rt sO rt r-i rt 00 rt 00 5 rt d rt J p 3 11 rj ri +’ r"^ 4i O 44 Tf 41 H rt 41 rt- +! rt 1 O G ■J d ri ri m frt frt ri d d 3 “ ri rf cr fr r^r —* ri d * »3 fO f"i n fi rr tr H rt rt - c- rn o ■rt — ri *A rt > rt ■ 1 " o O — o frt F- d — r ■ O O rj 'O cr. C in m fC 0 l-H > £ sd rt 4) kJ rt u w tl l> +i ri w 4i o O 4 ■rt 4i a o 4i rt d rt ■—■. r*-i 4H 4 rt j-. w - d — 1 — ri d ri — ri [ '"3 »“ h ■H II 41 H 4- + 41 L) O — 1 ~f tT c oc rr r i r: t; o d a sij d ri ri ri _] ri d ri 8 - £> £ *-• X -J rt; n v 0 'A v 0 q J rt CN rj rj ri n rl rt n G o c £ 4H c -H o 41 o rt C 41 A-? 41 4 a 4 X "0 Ll y C _3 K w « O' d d Ov d SC > < c (N ici ri vc re C — ■o X r-i in ___ ITi frt rj wrt frt «v U 5 " £■*% ■ti o II c ■fl o i- ri 4- i ■w -4 41 Q *=* 5ft p /*^S £ d II li o 4 d rtl Hi 4 d 4 d rtf X) rrt 7 O' ri O' T) ■o ■> i—i y rj O' ri o C C n (S (Z o u o d VC d d r vd fs J ri ri fn rrt frt rt frt d o ri 'A. ■4 o m r- VO X- V, X X rl a V\ rt 'C — frt n ■M 5li LJ w o d rj c ri o o d 1 d « sc p 1 / £ u c 41 O rt G" d 41 O' W* 41 d 4 rt rt rt- o d 41 cr o 4 H C 41 -4 41 't -H tn +i r- ri x 41 w 41 VC 41 in !U £ ■A J rtf frt rrt d d d (A d d jj- sc 0 ^ K c cr o Q w o d .— __ d o -=i K rs % 7 Tl K o r- O' o o O' S l/S f I d ■-• n vC c OC vc a vc in a o Sfj G d c w c Q c d o y c c -H 41 4! 4. 4 41 41 4 41 “ V y «/-j — JJv vc vC frt n 5? — mX d VC' urt d d d VD d d i-*-* u x rt m * 0 Q, at: 4J Q. !_■ r- 7 i d (3 X u |S c rt; d rt d K d rt o frt ri ir, O vO c r- d h A-r' 3 >k (U 4i ■ 1 Hi 4- -1 41 4 rt 4 4 r- V i*V W Li -V ** o a ^ 2 .« 'v rt r“ “> y S-X 1 -1 P _>T c i~ r- 5 *— V jl r- r-> o >k > r-* rt 5 r* r- _C jr rt £ M ^ g ■s S: c — g u ' TO r“ r* i—' SO •'— g 1 fn rt rt o -4 frt o -H r d 4i rt d 4i rt ri 4 rt d 4 VC c 4 q 4 *-* i — w o > 0 > s' rt 0 ■-■. o ■> T •d A 0 fjL X 4J y V oc ’/*! frt d rt; so r3 U' t> o JS vc d IT, d in d z r" 0 5* ** t/5 /’I w ? A w s Q > J^' IZ3 rt Vi rt LT. 4-1 rt c 0 0 S ^ •3 ^ \i ^ Jr r* £Ti O r/l o d C o o r* rt !U r 1 o >■ ^ S t r\ “3 CJJ c ri d rtr c irt w n o ._! r- d ri d 5 ■*—^ r Jl o >. o P rt Hh-» JJ 1/5 0 J s: ^ rt u c o -H C 41 [S -1 o rt m rt frt HI rt 4 r- 4i rt H frt 0 u > tr, o Cl Q V r/! ca ^ 0 - 1 CY ri frt n d n rrt frt rv -v y V ^ sC i 2 ^ r. ■■_! o I’’* 4-J CO Ff ri o ri d ri d rt d ri d i> d o o d frt d -s. o > *“ ■H ■1 H 4 rt 4 41 4 ■-«■ "■J - 4> y vc q frt vq q q q q vc !3 C i>i 1 --, 5S ^ ^ s ij — " ri ri ri ri ri ri ri ri t3 rs V, SJ ri -it fO =t *< ■-v sj ‘s -L a VJ S 5> fco ! a 0 ) '-M £ o ri m ■3 [X >. *_■ CL >i **v 0 g it « Q. at is Cl 1 0 rt 5n At "*«■» y ' '-s A | \J c ■> c r- ■b '«- r>4 5 T®» 1 ^ ,fc »i trt N SJ S rS dj r% 1 *>i V; sJ *>s ■Si L #\ 5u A sr _ vj d, +H a 2 K. *s T **m. Ak JC Cl r 5 1 <*> 1 ^1 “>bi Sj a> t“ £ 2 w L !>■ v> ‘5 «S* ■S v 5 G k. _y ■*. .bo *3 >■, > a K ■it I 1 >, mJ *»»* 8 -S 5f £J i*. ?T *1 5 SI L ? A r» 5 5 a 0 St t: S St -c u C 2 A ^ r j t> Vj !5 f-N- a G r "Sb ■XI a: l; ■« CL !-“ P r* A »-*■ L <: >- GJ ■r; S hi > a 0 V) > T *-r 'r X c. L > r* > >h d to X 14 Special Publications, Museum of Texas Tech University n. —Jiufo Xl ■LiARH .■''■t'to.'IFEJjWlfi.'JH t/tVihm SA'VtCJJ- ■Cth-irtTf^tliTt^ ■tl^OF I A>ll—Jarwfcr vurk^cila rf/rn'^ntu “’I" ttapafeimirptiFctm ffrri’ J wfl nJfr |" ■- ttujkiivmw grismi ^HfeftwSraa*H Hap*t*m*r *iw T> * jAftli * Pf**p iMiVjiTH-irjiffl,Xi« AS A I 10 —Ptoipfafr tctu fvrrU-1-t rliaifi’ma ' 41 r 1 nuwi rt^l '"^Fwfuthn uf tottenrailr *- Pn>pitk^’ift (tfflfOti Art-T i \i\ Jfiki/iicutriMiii* * KS * iJi ~*Prftplthrtw wrrvmxt .= 4j Avahi peyrtceaii lyp -?*' rliwta'^n r irnr.ir IJpf #3* + . fraiW tptckd m'nu -1 stVUki Hi AwUi iw mcrittitifTclili T¥ Li±Lci3l VehjILy Lcuilit (.IiiusiulI Specks IX'sIgfulkn Avuf/i tdnigtF . hYifrj r.M vijenlafi'i' ■AN.MJ \%-C'icri ilVixill;. Lemur lkl|L'.ll.ll SpLLlL- OctiUPl.lllUII Auifu «it e hit-uluhi. “■ 5 chiles tjpQfmr dnfeanMfiu'ft Figure 8. Neighbor-joining phylogram derived from the D-loop DNA sequence data from the 77 Avahi individuals with 26 out-group taxa. Species designated according to the distribution in the current literature (Mittermeier et al. 2006; *Thalmann and Geissmann 2000; *Thalmann and Geissmann 2005; **Zaramody et al. 2006). Values above branches indicate number of changes between nodes. Values within circles indicate support of bootstrap pseudoreplicates. Avahi catalogue numbers that are listed in Appendix II, but are not shown in this figure, are presented as haplotype information in Appendix lc. Andriantompohavana et al.— Woolly Lemurs of Madagascar 15 liL -ji'-K"—HuirA^TiiL* ■MttHlP— UpanAn r&fri =-U-UGI'—fikfliimHkVl rirth-hn J±- - ari — 8 ■ “^^■iS**** 1 C >m n eju. 1 ™ « = t tap&fcftlfS JhYli&t ■ I ' HA Ml Vi: —AtpHHi fitm an/. i> iu 14 "3* J ™ JtAAL. |Dr^-^ I ■ TAMP"^/ 1 ftOtt. "JV«« c"f*w*rfi‘ ■ TEAM - —-Pn^fckfcnno Airk»>*Yr -lit b| vi-—ipTf^prAkrcii* iLiiLhi ■/. jJEi >.■ -IS- HW ?™ £wus Arrirai riynurtn *'—AipfiAnilU' Uiwswwrj (hh: “1 Avahi jwyrtenni ivipc £) AvakS m-ridi/Mcrit? nrflhnw.iTJSiiHrrtJrftj .-h :iJji ffh.TtiiHtmifi* aieridio/mlii AvijJu vptNrm; M.y 41 .■UiMy fmwiT .illtnhi iJ'Jii Y.J'iir MAH -hid 1*11 1 Cfcwart MU I ICu jjliuAr i.s iJiir.Miui'rL Figure 9. Maximum parsimony phylogram derived from D-loop fragment sequence data from 77 Avahi individuals (one of 144 most parsimonious trees). Values above branches indicate number of changes between nodes. Length = 1,271; Cl = 0.5382; R1 = 0.8786; RC = 0.4728; HI = 0.4618. 16 Special Publications, Museum of Texas Tech University ftm urn //- " : MUracebux rtiwiitheiisix -- Microcfitus rufitn 4 i s.j« i ^ _ c;ar« "" r Cheirogateta mtxffw -MRAN0229 — CfeirtMtifatf majur nfli 1 ft|5| * ti.24* q m ■HAN2l-'“" t l‘&red , 0 variegata variegate ■RAW043- Puiemur fithtia rufm Y"tiapatomtr griseus occidcntaUs 0.4M5 iiijjhlE/ Al RANol 38 h' Hapalrmur jimtis Ifiipdhuttw grisetix g riwux *912 Ql43» ■■■ fiapafemur mtwtts ■ANAEpS .. l.epilemitr unktiFtmimsis rJ.-T.K v^s—\ ' turfs/ fruit's QuO «i.d]5 u lHM ^ ®^PRAN0132- Propilhecus edwardri Q.ixi'^hIt-TaRJ 4 PstiifitfiMts latkfUhti ‘ii tasiae.io pemeri t £? 7 ^ q | i PrttpiitH'vfvi diadems 0.SffrBOR2.. k'n-tpsthecitS cotfwtvii ' UejaMJ .—Propilhecus mursatii — Prtspifhectts tlwkrni stnkvffi ■■ Pmpithet'its rfeokem co.miiaftts Pfioptthecta wmrarixi O.lKS aixijrBEMA4' fAM4.7~ .MlH >MOR££ ■■■■ o.ots u.rtG£ ■ft Oil B.I40 Avahi fieyiierusi type #2 Avatii meridfomtlis raman&rttso&vana Avahi merklionalis im’rhfioruilis 'KfiSSm Avahi peyrierasi typ& U7> Avahi species nova #1 0»AI>. M’ialh ft.006 c-iAmis Kr J i 'lid L i| TAD4 :5 g *1 T:\IM.9 Avahi laniger MIZAIO ii /mi If “H NAKaSPiI ■NARA4.IQ KOM unicotor DjOftlla W3 Avahi cleesei mtfa O.-OiKS LUMiJ Avahi occidentals 0,05 substi l uti ora/site Figure 10. Maximum-likelihood phylogram derived from D-loop sequence data from 29 Avahi haplotypes. The phylogram presented with branch lengths proportional to the number of changes (values specified on the branches). We obtained the maximum likelihood phylogram (-In likelihood = 6398.15) from the D-loop alignment from a transition/transversions ratio of 2.28 (k = 4.66) and y shape parameter of 0.54. (FAN6.6 and FAN6.7 are identical to haplotype FAN2.14; FAN6.9. FAN6.10, and FAN6.11 are identical to haplotype FAN2.15; these individuals are not displayed in any of the figures). Andriantompohavana et al.— Woolly Lemurs of Madagascar 17 n OK T4 V/~ jjr 121 l«5 ITT i£L il* ion u “7T .ANKT -“Jtf/frecc&tw mRMwr^ -RAKO2S0 MicFCKthas nifits Ok — —-iwipmuo --lum — 1 i i ABS - - C/J^ragtrfr'iM mewl*? . ., RAN022fl—»■ CArirtOj'ii/m.sJ jfnj/cw anailj —— j^fuirrtutr (mknirmensis v> ■VK ■OK yA lt, , ■w ]lOkf*n 'Prvpiihtrcus vemauxi ICO L£i -BAWWL^ ICQ FAN 21 """-Eiitcnmr fuivw; ntfit.% —ttqpatwnvr g r grtievs ^-'-Hitpafemur g, occidentals QAR9 1W ICO .M I no ira .4vahi peyrierasi type #2 w ]?: IOG 100 SL ICO S'). ]£_ [17 Avahi nteridianafis ranmmmtsoavana '“pi?, mm Avahi meridbnafts meridionafis u M R2 UK , 2J Hr t)V; I? \\f>67 \N( ANOJM K \ Nt 13 .'I i fan: m J’A\2I5 IAN? 14 r\S5 51 i-w ;i> AC >:,:■> M 1/ \ if M / \ ;■ Ml/. AM iTADJf- LTAD4 i , J4V Ml/M ; NAKA4.I NA|?A4.2 t AkA-t.M \RA4 J) aka i ■ AKA-i Id JARA-l.ll \ARA-I 1“ SARA I \> NAHA4 .M Avahi peyriurasi type #3 Avahi species nova U I iTAL %x I-.- 4 -Ml.' ' F r Vi h.m Avaki ftmigvr «■ _ !CO LL ANT5.S AMJ.'i Zp rtMi m & — HK’tisinsje* 111 MAS 111. MAO lil-MAI2 ™,fsfmalj liFtKVlA 14 UAKN P®. aigj MARS* MAR*-ft M.AKM KT i — M urn Avahi unicolor Avahi citesei Avahi occidcntalis Figure 11. Maximum parsimony phylogram derived from PAST fragment sequence data from 77 Avahi individu¬ als (one of 53 most parsimonious trees). Values above branches indicate number of changes between nodes. Length = 3,551; Cl = 0.4816; R1 = 0.8721; RC = 0.4200; HI = 0.5184. 18 Special Publications, Museum of Texas Tech University psvslobttisls ANK 7''' __ ^^-'Microcebta rvfus CheiTvgtih'Us ftittjw .. jtwjar ANAl /- Lppitemar unkmmiemij; fe* I *v 1 III " 1 ' -vL)^ ?. Hapakrtmr siffttts r. Hapalemiir aureus ^ cj. griseiix ,2\\ — HtifitiU'inur g. arcAfetfftaftr “ —£rrA»nur futvus mfm " Ibft'cHi mrtegata vsricgata " 1 “ i? i pi! r" 11 ‘5 Y 1 cj4s r.toimt "Ptvpithtcus ec/Hur^i IRAK 046 Pm/Wf/lftt/.T L'i.VWdJJdJl'l 1 " I MQ fcHO oil MU OOHIf, Avahi peyrierasi type H 2 Avahi meridionaiii ranianantsoavana ; Avahi merldiotialis meridianaiis IRANI. :r.’ I '•; ■ ;■'; . ( A vahi peyrierasi type ff3 1 AM 13231 -i an: 14 Avahi species nova # I i.i.wi «ID 0(01 pBl Tvni^ ['All 3 7 rAw.zr) Ml/.A, 10 MIZAI2 MIZAM *Diin2 GW* urip^ JTA:|>4.9 : ,.0^\1IZAI3 NAHA4 ! N \H \4 } NAK.A4 |3 NAHA4 •! '• MARA-1.12 SSKS:W NARA4. Avahi iaitiger (ym n m mi Jf KlAM24 = , _Jgj ^ n jj simiLS _Jiil_rftAfflH 52 *—Hapaltmur timcit? m RANTMI ITS i?-— Hapatemurg. griseuf 219 UiAS-il 2 23!““ Haptdermtrg, occid&italis ■■MANtMS —Euimmr fiih'sm nifux m LW 164 L31 M | j ii ■ "Ei MIZAJ.31“ " JndA intfri ■fan? I—— Varecia variegata — ANALi -. Lvfiitemur ankiirvttwnsi* I2K ■RANUflE. Propifht'cua cdwartisi -MoRW""' Propiihecm vcrrctntxi 2lfi - JOghjin^fH J u ■— Vj ■5 a E v> —■ tu 1KANCM4. JVOP5.H t’243 _ LiRAMtni'JH m Avahipeyrierasi type J^3 Avahi meridionalis ramanantsoavana Avahi merklio/iaiix meridionals Avahi peyriemsi type l >2 Avahi species nova U 3 ji IWOl i .TADS X ™» M BTAD37 Imizaio ri J-T,\DM f, -TAD4.3 — SlTAIM.tf JTAUJ. 9 “MiZAO TffNARjM.I nplNARA4.2 IIt/naRAAIG MsARAAII Avahi ianiger n Western Avahi species M; 29£ANT5,8 ' ANT5-.I0 Aft f &EMAK rnnililMA4 Avahi imicohr Avahi cleesei 111 “lilTMAU ISmarIs Avahi occidentals Figure 14. Maximum parsimony phylogram derived from the D-loop and PAST combined DNA sequence data from the 39 Avahi haplotypes with 19 out-group taxa (one of 20 most parsimonious trees). Values above branches indicate number of changes between nodes. Length = 4,999; Cl = 0.5141; Ri = 0.8392; RC = 0.4314; HI = 0.4859. Andriantompohavana et al.— Woolly Lemurs of Madagascar 21 Table 2A. Diagnostic nucleotide sites from the D-Loop Pairwise Aggregate Analysis (PAA) of Avahi . ___ 1111111122222222233333333333344455555555 1260155799927777889900011333349902900002244 6046289245975679785602934234686712925671538 RAN06 6 ATCGAAAACACAGGAATTTAAGTCCTCTACAGCCAAAAATCGG RAN0319B . .c. RAN03 4 8 RAN02.10 V0P2.13 VOP2.14 V0P2.15 . .c. RAN067 . . . ."TC.. ..,T... . T . G . .. .. . . GG.C HANOI 9 9 ... * ^TC. . ..,T... . T . G. . . . . . GG.C RAN0320B ....TTC*. ...T... . T . G. . . . . GG.C RAN0261 m ....TTC.. . . . T . . . .T.G... . . GG.C I486 ..G."ACT. , , . T . . . .T. . .GG. . . T. . M89 ..G.TACT. . . . T, . , . T. ..GG...T.. M1G7 ..G.TACT. ...T... . T. ..GG...T.. Ml 08 ..G.TACT. . . . T , . . . T. ..GG...T.- Mil 9 ..G.TACT. . . . T. . . .T. ..GG...T.. M150 ..G.TACT. . . . T. . . .T. ..GG...T.. Ml 55 ..G.TACT. . . . T. . . ,,T. ..GG...T.. Ml 59 ..G.TACT. . . . T. . . .T. . . GG T . . MAB4.8 ..G.TACT. . . . T. . . .T. MAB4.10 ..G.TACT. . . .T. . . .T. ..GG...T.. FAN2.14 . c. ....TTC.G . . T. . . . . . . C. .TC.T.. . .GG. .C. . . FAN2.15 .c. ....TTC.G ..T.... . . .C. .TC.T.. ..GG..C... FAN2.19 .c. ....TTC.G , . T . . . . . . .C. .TC.T.. FAN2.2 0 .c. ....TTC.G . . T . . . . . . .C. ..TC.T.. ..GG..C... FAN2.21 -C . ....TTC.G . . T . . . . .. . c. ..TC.T.. ..GG..C... TAD1 ....TTC.. C . . T . . . T . .GG.... A. TAD 2 ....TTC.. C . . T . . . . .T. . . GG .... A. TAD 3 . . . ,TTC.. C . . T . . . . . T. .. GG. ...A. TAD 19 ,...TTC.. C . . T . . . . . T. , , GG . . . . A . TAD 3 6 . .G. .TTC.. C. .T- - - . . T. . . GG.... A. TAD 3 7 * ....TTC.. C..T... . . T. . .GG.... A. TAD4.3 m m m .G..TTC.. C..T... . .T. . .GG.... A. TAD4.8 * ■* * .G..TTC.. C..T... . .T. ..GG....A. TAD4.9 ■ 9 * .G.,TTC.. C..T... . .T. ...GG....A. TAD4.2 0 ....TTC.. C . . T . , . . . T. ...GG....A. MIZA10 ....TTC.. C..T... . . T. . . .GG.... A. MIZA12 ....TTC.. C . . T . . . . . T. ...GG....A. MIZA14 ....TTC.. C. .T. .. . .T . . . . GG.... A. MIZA13 .G..TTC.. C. .T. .. . .T. . . . GG.... A. 22 Special Publications, Museum of Texas Tech University Table 2A. (cant.) _____ 1111111122222222233333333333344455555555 1260155799927777889900011333349902900002244 6046289245975679785602934234686712925671538 AND4 G . T , G * GTTC. .T. .T. . ....T.GGG AND 5 G . T . G . GTTC . .T. .T. . ....T.GGG AND 6 G . T . G . GTTC. . T . .T. . ....T.GGG AND 11 G . T . G . GTTC. . T . ....T.GGG AND12 G . T . G . GTTC . . T . .T. . .T.GGG AND 13 G . T . G . GTTC. .T. .T. . . . . .T.GGG AND 14 G . T . G . GTTC. . T. .T. . . . . .T.GGG AND 19 G . T. G. GTTC. .T. .T. . ...,T.GGG AND3 3 G -T.G. GTTC. .T. . ..T . . ,...T.GGG AND34 G . T . G . GTTC . . T. .T. . .T.GGG NARA4.1 . TTC . .C. .T . .T. . .GG . NARA4.2 . TTC . .C. . T. .T. . .GG. NARA4.10 . TTC . .c. .T. .T. . .GG. NARA4.11 . TTC . -C. .T. .T. . .GG. NARA4.13 .TTC. -C. . T. .T. . .GG. NARA4.17 .TTC. .C. . T. . T. . ......GG. NARA4.18 .TTC. .c. . T. . T. . .. .GG. NARA4.2 3 .TTC. .c. . T . .T. . .GG. NARA4.31 .TTC. . c. . T . . T. . ......GG. NARA4.3 2 . TTC. .c. . T . . T . . .GG . ANTS.8 • . .A. . . TTC . . c. . T . C.T . . TG . .GAT..GG. ANTS.9 * . . A. . . TTC. .c. .T. C.T. .TG. .GAT..GG. ANTS.10 * . .A. . .TTC. . c. . T . C.T . . TG. .GAT..GG. ANTS.12 . . . A . . .TTC. .c. . T. C .....T..TG. .GAT..GG. BEMA8 • . A . C . .TTC. . AA . cc ...GA..*CTG. .. C ...GG. BEMA9 ■ ,A,C. .TTC. .AA . cc ...GA...CTG. . * C , . .GG. BEMA12 • . A. C . .TTC . .AA . cc ...GA...CTG. ..C...GG. BEMA13 - .A.C. . TTC . . AA . cc ...GA...CTG. .. C ...GG. BEMA14 * .A.C. . TTC . . AA . cc ...GA...CTG. ..C...GG. MAR2 9 . TTC . -C. . T. . CG.T . TG . ..C..GGG. MAR4 6 .TTC. .C. . T. .CG.... T .TG. . . C. .GGG. MAR52 .TTC, . c. . T . .CG....T.TG. . . C . .GGG. MAR54 .TTC. .c. .T. .CG.... T .TG. . . C . . GGG . MAR5 5 - .TTC. . c. .T. . CG .... T . TG . ..C.. GGG . MARS 6 .TTC. .c. . T. .CG....T.TG. ..C.. GGG . MAR6 0 . TTC. .c. ,T. .CG....T.TG. .. C..GGG . MAR61 . TTC . . c . . T . .CG....T.TG. ..C.. GGG . Tabl e 2B, Diagnostic nucleotide sites fro m the PAST Pairwise Aggregate Analysis (PAA) q/Ava hi. Andriantompohavana et al.— Woolly Lemurs of Madagascar 23 o\ oo o\ \> ch vo a\ in a\ lo Ch LO ov, a> ^ Ch co ov co CM 0\ CM 0% i—1 OV iH CO CO CO 00 CO CO CM O 00 r- oo> t> vd t> o r- o [> o vo co vo 0 VO CO VD CO LiO CTi m i> LD l> ID in cm o o co co co co co CM CO CM CO CM l> cm m (Ml CM i—l (H LO i—I CO rH CM t— t i—! i—I i—I i—I i—I rH O rH CD o\ CO \> LO CM L0 LO LO CM CM 00 0 CM LO O CO in co o VD O O co LD VD L0 CO CO M 1 r-i CO i—! m VD o VQ CM O CM i”t o m VD <—I m o\ co VD CO in co i—i 00 ■sF I—I 00 CM CO LD LT) l> o < 0 H < U < u 0 < Eh 0 a a < o o 0 Eh Eh Eh U 0V VD CM 00 1-1 I-1 1—1 CM VD CO CO CM * * > 1 ' VD r-i CM CO X ■Q X) Xi X! O O O o CM CM CM o o o o X) r- 00 av o L0 CO M* sP CM 0 CM CM CL CL CL o o o > > > vocnoo^HinmLncQaQ oo co i—it—ii—it—ii—ii— &L pLj pL| 24 Special Publications, Museum of Texas Tech University 03 00 LO o\ l> CM CT\ VO CM 03 LO 00 o a CJ u u o u a u u m LO \> ■ o o o o o O O O O O o o d d * G\ LO CM * Eh Eh Eh Eh Eh Eh H Eh Eh m LO u o o o 0 o o o d d d d d o u 0 0 O a o a O O O O ov O c\ CO CO m CO LO H Eh Eh Eh Eh Eh £h Eh Eh H o> CN 03 o\ CM O < m s—l VD m s—1 o 00 o < o o O O O o O O O o CO CO CD CO CO LO- CO CM VO o 00 LO 1 CO CO' i> VD !> O CO < < < < < < < < < O O *sP O o t—1 VO a\ CO h Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh U< Eh U< Eh Eh Eh VO r- rH • < < < < *< <1 CM 03 : rH 'Np u a u u a O a U 0 O u a 0 a a u u u u u u u a u u CO VD O cm m • ov vd [■"- Q Q Q P P Q Q < CO LD l> CO ro CO i> o 00 i r- o i> o 1—1 CD co CO CD i> X-1 CD CD LD CD CO CD in a\ O ld O CD LD i> CM LO O in CM CM ld O i—1 LO O O ro CO CO CPi ro CO LD CM co rH CM co Cft CM r~- CPi CM ID CO CM CD CM i—1 CO rH LD LD r { CO ro i— 1 CM 1—f T —{ i—1 CO 1-1 t—! 1 - 1 T—1 1—t H o 00 i — 1 o CM co CO 00 LO o LO in -sF M 1 o CM O i—t 00 CD * * ■ * * •■ 0 000000000000000000000000 ■ * uuuo<]iCOO\HHHO^lDCN^^lD LDLDiDiD<<£<* ^ &\ CO o\ d o> r\] 0> CM CJ\ rH tH CO ^ co d oo d LD CM CM cc d CM ID O 00 LD 00 o VD O d OO ID •4 ft ft P o o * 4 * * U U H H co d ID CM LD LO'U U d 0 0 o\ ^ u u oo 00 O rH VD VO CM CM CM CM CM CM CM CM CM CM CM CM CM CM (—I <—I t—I CO ID LD CO CD ri iH ID ro tH cl co t—l d i—l H CM CM <—1 i—I co i—It—I i—! O D LO O LD CD o in d d CM o o d o O H d oo vo o cn ^ vo 00 CO VD co d 00 d 00 VD CO ^ co d d CO d ID CM o <—I o\ 00 CO H H H i—! O d h H CM i—I t—I d H i—I M* O d O VO ID LD O d s; h o id d o .Vi 4 ID CM CM * * rH O CO H fe * 4 4 4 0 0 0 0 ID O rH * * rH o d Eh 4 4 4- ■4 LD o o < < i— \ d O') d 0 4 4 4 ■ 4 4 ■ * d St 1 0 0 rH d o\ o < It 4 4 4 d d o\ » ■ rH d 00 Eh 4 4 4 * 4 d d w * ■ f—1 d CO rH < 4 1 * 4 4 * 4 W V CM o> X—1 Eh *—\ d VO d 0 4 ■ 4 ft CM co G\ - rH d ID d 0 * 4 4 4 0 0 0 0 CM d G\ . * i '—^ d rH Eh 4 » • M 4 0 0 0 0 CM D CO • ■ rH d d o\ < # * 4 CM vo ■■ ■ rH d d 0 0 * 4 4 4 CM x—1 d . » rH d d d H 4 4 4 4 ■ ■ m H LD ID Eh Eh rH CM d o^ 0 m 4 4 T—1 d d . ■ t—i CM d d 0 4 4 1—1 CM i—1 0 0 rH CM d 0 Eh * 4 4 4- 4 1—1 I-1 co k * x—1 CM vo rH Eh * 4 T—1 rH 'st* * * x—1 CM x—l d 0 * 4 4 T — 1 t— i [—1 i< < (—i rH vo co CJ « 4 # 4 4 4 4 rH O co H H rH x —1 d CJ\ H 4 4 4 < < < < i—i o CM Eh Eh x—i x—1 d CO Eh 4 4 ■4 CA d * * rH rH d LD < 4 4 4 4 4 4 4 0 H 0 U Eh H H Eh < 0 O o E-i H H H Eh H H H E-| h h H < < < < 0000 0000 Eh E- Eh H Eh Eh H H 0 0 O O O) CO x — i no LD CTi T — 1 o vo T . —1 « T—1 i—i <—1 d cr> vo CM vo d d CM * * * vo i — 1 CM d O O O O CM CM CM O 0 O 0 P-t P-i P-i O O O > > > Andriantompohavana et al.— Woolly Lemurs of Madagascar 27 CM ro un LD eg ro ro T— ! CM rH LO CO CM rH CO CO CM :—\ ro \ —1 CM 1—1 CM CM CM !—! rH VO CM rH i—1 rH CM o CT» VO CM o LD CM o LT) ro CM o CO CM CM o CO G CM o rH CO < —\ CO VO o x—l o\ VO ?— l CO 03 VO tH CO l> CM I—£ CO O O T —l CO VO rH T— E 00 MT oo T—1 CO CO r I—1 00 CO ID l—1 CO rH CO T —II 00 1-1 CO f—1 CO O o I—1 r- 00 VO rH r- o 1—1 rH r- o 00 T — 1 CO T — 1 CM l — 1 LO 00 LD rH If) CO rH in LO CM i—1 LT) CO O i —l in CM LD T — 1 id rH av 1—1 O r- rH LTj a\ x—\ in rH o CO i—i o I> x —1 CO av r~ i—1 CO av o x —1 ro 00 x —1 CO a) x —1 x — 1 ro vo CO X — i CO (JO CO X —1 CO 'M 1 !-1 I- \ CO ro m rH CO ro vo rH cn ro co rH CM O CO e—1 CM O CO T—1 CM O o 1-1 CM VO l—1 x —1 CM rH CO X—\ rH VO 00 rH rH CO CT\ i—1 i—i CO CD rH i—l CO LO rH i—1 ro i—t rH i — 1 'CM hT x —1 i—1 1 —i t" t —| x —l X —) O 1 i—1 O o LD [—1 O VO O iJ 1 t— 1 o in r- 7a6/e 2Z?. (coth x — 1 o in CO EhEhEhEhEhEhE-^EhEhEhEhEhE - 1 i Eh EhH h h Eh H H E-1 E—f E— 1 E— 1 ■ **#*■•«■ ■ « • » * | « 4 c t * l i * # • # uouooouuuuouuoouuuuuouoou EH £h eh Eh H H Eh E-h Eh H HH^LhHHHHEhH HHHHHHHEhEhEh Eh Eh Eh Eh R ^ H Eh Eh H H H E -1 E h Eh o O 0 O O Eh Eh Eh Eh H Eh H Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh Eh EH Eh * » a U U U U a o a U ■ U 9 9 < rii < < < < < < C < < < c < c < < < < < < < < < << 0 CO 0 LO i — ! CO CO CD u 0 o 0 0 0 0 o o a 0 0 u 0 0 U 0 0 0 0 0 0 U 0 U 0 CO 0 CM it < < * A O 0 CO 0 O < < < < < < < < A rH CO CD 1-1 * A * x — J CO 0 GO * A 0 x — i CO CO r- * A < 0 CO CO no jjn H H 0 0 0 0 0 0 0 0 0 0 0 0 Eh 0 0 0 0 H 0 0 0 0 H CO l—i co 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 rH CO T-1 ro It rH 00 O 0 rH 0 CO CD * rH 0 0 rH . » • O CD O 0 o 0 0 O 0 0 0 T—1 0 o co O U U 0 * 0 0 0 0 0 0 0 0 U t rH CD rH CM ¥ * 0 0 0 0 0 0 0 [h 0 0 < tH L0 CO LO rH no CO 0 *—$ m no CM •* < < < < < < rH LO ro O < < < < < < < < < < < < < < < < < < < 1- \ m CM no H E-* H 0 0 0 Eh 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 H LO 1-1 00 ¥ i—i 0 0 0 rH 0 no GO i ■ - * 0 0 Ifni 0 0 0 0 0 0 0 tH 0 no 0 m A * 0 i —\ 0 o 00 * T - 1 0 o 0 < rH CO GO 0 * 4 0 0 0 0 0 0 0 0 0 0 1-1 CO 00 o ■ 0 0 0 0 0 0 0 0 0 0 A T-1 CO CO 0 A rH CO CO 1—l !—1 CO CD CO 0 0 < l—1 CO no CO o 0 0 0 0 O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T — 1 CO 0 ?- 1 u U U 0 0 0 0 0 0 0 0 0 U 0 0 0 0 U 0 0 0 0 a 0 0 rH CO ro GO * 0 0 0 0 0 0 0 0 0 0 A rH CO CO CD 1 * ■ » ■ 0 0 0 0 0 0 0 H 0 0 *— 1 CO co ro 0 CM IT- GO 0 rH CM 0 ro rH CM 0 o ■ - ■ 0 0 0 0 0 0 0 0 0 0 *— \ CM CD T-1 1 rH CM rH CO r—1 rH l -1 l -1 CD CO 00 GO rfj < < C < < < < < < < < < < < < < C < < < < < < rH I-1 CO CO 0 i—f T — 1 co no rH 1 — 1 co <— 1 ■ • • * o 0 0 0 0 0 0 u 0 0 rH l —1 CM 0 u u O 0 0 0 U 0 0 0 O 0 0 0 0 0 O 0 U 0 0 0 u 0 0 rH T — 1 <—\ 0 0 0 o 0 t — 1 T-1 i—i 0 * 0 0 0 0 0 0 0 0 0 0 i —\ O 0 in ■ rH O CD O § 0 0 0 0 U 0 0 0 0 0 A ■ t—1 o LO o H o LO ro ■ o rH co 0 co ro x—1 D3 l — 1 0 l — 1 b—( rH \— 1 t—1 CM co CO o CM co 0 00 tHr-It—li—t ! i—1<—i(—lrorOi<^<1rt!i<[I i—i o CO VD T-1 o 1—1 r—1 \> o 00 rH VD rH' cm rH m 00 m rH LO 00 rH LO m OH rH m 00 r- rH in CM LiO i—1 L0 rH OH i —1 r- r- i—1 in CO i—1 tn H-* i—1 o CO i —l 'Sp o r~ i—1 oo OH r- i—1 00 OH o n—1 00 CO i—1 00 CO T—! <—1 00 VO 00 i—1 00 in 00 <—1 00 XP I 1 <—1 00 00 OH i—1 00 ro VD i—1 00 ro 00 i—1 CM r oh i—1 cs r ro i—1 > . ilAlllJ fhxh ■■*• ■) -IK4L2 tn ittpnVrii -15 i,R4 2 i ,l .!-ri«'i'r l T li^i lulTiV WiVi ~ BI:MA4- Pny fa fcwcitf tffatan *Epr4i7d -M\M 7 /Kn-.fn fhm u* *T r mxjw.irtvi -MC:■ ftuK/V.^v 'Wvmt r*m tiuw ry[K -2 mi r.i,r,jii..i!,'Mr.vi.jm\i ft! ItTffiA'rtHIltit jww^mMjpc m3 .-|r. 49 «■ t +M 49 T>- 9C 49 99 109 W JHh K4 41 Lihi TJ ■W IIHI JlEL IH >lXl 71 IH IDG 94 49 JtiL J3L AM&L7- MicrtrCvbiti MPpl&bentfa RAN0250- Mknit^fytis rujux (JAR*- 1 —- Chemigatcmt meatus RANQ229-- Chcirogafeux major asaO—-- Lepiitww airtofFmewts k BilUlU — ftufkitc-untr g. gri.i S jiapatemur g, occidmlatis ' ■ Jtapatvamr g. giis^a i CARP 'ANALZ.23? RANtHS. Evlctmrfafvti* ritfiis 1 RAN2 Vtrrvt'hi Yitrttfgalu vfirh’gvlf* RAN03J2 '" rntfAthwiu- - ■ mprwc-. Rmittihccua wrremai 99 —* •w Asufii pQ'riemtt lypc 41 Awthi m. ritfHiinanisoavQwi ■ RAN067 “ RANDIW - HAHOSAl - RAKO320 - y.wl i-s -IAVMS -■ F AN-’ IV - I AN2.21 -* IAN2.2U - r.Alil - TAD2 - DAlii ^TAOl^ - TaIIJ? -tajdi.io -MIZA1Q ’MlZAii -MIZaM -* jALJlt ■* IAI14 J -Taijj.h - IAIJ4.V - M3IAI3 -NARA4 1 - NAKA4 2 - VaR AJ 1 1 - NAKA4 2i - N \KA I " -NAKA4 hi -NARA4 li -NAHA 4 17 ^NARA4 IX _ NAHA4, *1 J A NTA.fi - avtj.LS -BEJrlUi -IIFMA'J - BEMA12 - BE MAI 3 -BLMA14 ■ MAKS' , MAR4* - MARtt MAR'S , MAR64) ■ MAR 54 MARSrv 'MAR* I jIiwAi ffl. meridional^ Avahi pcyrk'msi lypc Avahi sjwcics nmu #1 Avahi Iwrigpr Avahi tmicaiar Avahi cltesei Ai-abi (A X'idettiaiit Figure 19. Bayesian majority rule maximum likelihood analysis cladogram derived from the PAST DNA sequence data from the 77 Avahi individuals with 19 out-group taxa. Values above branches indicate support of bootstrap pseu- doreplicates with values greater than 50% reported. 38 Special Publications, Museum of Texas Tech University 2 < L- 1 o s. < C4 ;l a. ? 1 m r kl £ V r- t _r < " ■tt; D =_ £ i § 5 < o L J a £ ^ t V ti =r -*3 5 “1 c ■s -Jf' 5l it ft < £ < z; < £4 5 s s < T & ^ -Hi, s< > 5" > £ S U 5 ■§ ^ £ is 5 & S] ft < : § J £ ! u T N T E «a 9 -f )< flB v\ t£ < 5 Figure 20. Minimum spanning network of Avahi haplotypes calculated using Arlequin Version 2 and Network Version 4.11. Identification numbers denote haplotypes cor¬ responding 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 the circles approximates the number of individuals with matching haplotypes (circles without any number represent one individual). Andriantompohavana et al.— Woolly Lemurs of Madagascar 39 Figure 21. Avahi meridionalis, southern woolly lemur, at Andohahela National Park (Manangotry). Photo by Edward E. Louis, Jr. 40 Special Publications, Museum of Texas Tech University Figure 22. Avahi ramanantsoavana, Ramanantsoavana’s woolly lemur, at Manombo Special Reserve. Photo by Richard Randriamampionona. Andriantompohavana et al.— Woolly Lemurs of Madagascar 41 Figure 23. Avahi betsilio, Betsilio woolly lemur, at Fandriana District (Bemosary Classified Forest). A. betsilio was formerly referred to as Avahi species nova# 1. Photo by John R. Zaonarivelo. 42 Special Publications, Museum of Texas Tech University Figure 24. Avahi betsilio, Betsilio woolly lemur, at Fandriana District (Bemosary Classified Forest). A. betsilio was formerly referred to as Avahi species nova #1. Photo by John R. Zaonarivelo. Andriantompohavana et al.— Woolly Lemurs of Madagascar 43 A comprehensive dataset that includes repre¬ sentatives from multiple populations and sequence fragments for all recognized genera and species will be required to conclusively resolve the taxonomic relationships between the family Indriidae and other genera, but this data does offer corroborative evidence for establishing species versus subspecies status for regional populations of the genus Availi based on the phylogenetic species concept (Cracraft 1983). For in¬ stance, Zaramody et al. (2006) suggested that the status of A cleesei was questionable based on a relatively low genetic distance between A. cleesei and A. occidenta¬ ils. By evaluating the uncorrected genetic distance, morphological, phenotypic, and pairwise aggregate analysis data, along with the inclusion of the third rec¬ ognized western Avahi , A. unicolor , we conclude that the parameters generated with the multiple data sets are analogous to those established with other species, including comparisons within the genera Propithecus and Lepilemur (Figs. 8-14; Tables 1-4; Appendix Id; Mayor et al. 2004; Thalmann and Geissmami 2005, 2006; Andriantompohavana et al. 2006; Louis et al. 2006a, 2006b). For example, there exist significant differences within the cytogenetic, phenotypic, and morphological data among sifaka species which con¬ sequently led to full species status for seven sifakas of the genus Propithecus (Mayor et al. 2004; Mittermeier et al. 2006). Despite the elevation to full species status for the sifakas, the genetic distance based on D-Loop sequence data between Propithecus tattersal/i and P. coquereli or P. edwardsi and P. diadema is relatively modest (1.79 and 1.96, respectively; Mayor et al. 2004). The minimal genetic distance between individual sifaka species, however, can establish supportive evidence in favor of the legitimacy of A. cleesei since the genetic distance between each pair of sifaka mentioned previ¬ ously is less than estimated between A. cleesei and A. occiclentalis (3.86) and/or A. unicolor (4.17; Mayor et al. 2004). Despite several citations which identify type locality of Avahi laniger in the Retanimena country, An- tongil Bay. Maroantsetra, the type locality of A. laniger remains unknown or uncertain (Jenkins 1987; Groves 2001; Zaramody et al. 2006). Nonetheless, based on phenotypic and molecular data (PAST fragment only), two types of A. laniger from the extreme ends of its range are evident within these data (Figs. 1,4, and 15; Tables 2-4; Mantadia and Mananara-Nord National Parks). Further research, including comprehensive samples and data generation, will be needed, however, to conclusively resolve the taxonomic relationships of the woolly lemurs between the Mangoro/Onive Rivers and Anjanaharibe-Sud Special Reserve. The data also established two types of A. peyri- erasi (Figs. 8-14, 16-17; Tables 1-4). In forest frag¬ ments south and north of Ranomafana National Park (Tolongoina Classified Forest; Zaramody et al. 2006), unpublished data reveal that both types of A. peyriercisi are found within the same habitat (within Vohiparara, Ambatolahy Dimy, and Talatakely, all subpopulations within Ranomafana National Park). However, no be¬ havioral or hierarchal data exists within a population or family group, so further research (mitochondrial DNA sequence and genoty pe data (pedigree analysis) combined w ith long term behavioral observations) is required to establish the relationships between these two types of sympatric Avahi. Furthermore, we suggest that the two subspecies, A. meridional is meridionalis and A. m. ramcmantsoavana , described by Zaramody et al. (2006), should be elevated to full species because the molecular, phenotypic, and morphological data is equivalent, if not more conclusive, to the other previ¬ ously recognized woolly lemurs (Figs. 8-14, 18-22; Tables 1-4). In this paper, the current Avahi taxonomy w as ex¬ amined according to the Phylogenetic Species Concept (PSC) sensu Wheeler and Platnick et al. (2000), Mayor et al. (2004), and Louis et al. (2006a, 2006b). The diagnostic characters or attributes define evolutionary significant units (ESUs). Several authors suggest that ESUs are equivalent to species as determined through the Phylogenetic Species Concept (Cracraft 1983; Barrow'dough and Flesness 1996; Amato et al. 1998). The constant addition of samples to the PAA data set will continue to test the distinction of these characters. The identification of a new species in the following description establishes the essential need for extensive as well as detailed sample collections across Mada¬ gascar to determine geographic ranges and taxonomic status for all of the woolly lemurs. This research is especially needed on opposite aspects of all rivers (for example, north of the Mangoro River to Anjanaharibe- Sud Special Reserve). Due to the inability to maintain woolly lemurs as long-term live vouchers in captivity, whole blood, morphometric, and e-voucher photos will 44 Special Publications, Museum of Texas Tech University serve as the type series for the newly described species, Avahi species nova #1, described below. In this case, an attempt was also made to identify existing museum specimens to represent the type series, but a museum specimen has not been identified from this specific region which could serve as the holotype. Avahi betsileo , New Species Type Series. —Whole blood for FAN2.14 (TK 125757), adult male; FAN2.15 (TK 125758), adult female; and FAN2.20 (TK 125759), adult male; are stored and curated at the Museum of Texas Tech University. Individual measurements, e-voucher photos, and collection data are given in Avahi Field Data Appendix la. FAN2.14, FAN2.15, and FAN2.20 were collected by Richard Randriamampionona, Jean C. Randriamanana, Gerard Nalanirina, Razafindraibe Jean, Rambinintsoa Andriantompohavana, and John R. Zaonariveloon 13 July 2002, 13 July 2002, and 15 July 2002, respectively. Type Locality. —Madagascar, Province de Fianarantsoa, Region AmoronT Mania, District of Fandriana, Bemosary Classified Forest, 20°20'60.1 "S, 47°33'36.1 "E, and south of Mangoro River. Description. —When A. betsileo is compared to other southeastern woolly lemurs, Avahi betsileo (1.05 kg) is approximately the same size as A. pey- rierasi ty pe #2 (0.86 kg) and A. meridionalis (1.06 kg), but slightly larger than A. peyrierasi type #3 (0.98 kg) and A. ramanantsoavana (0.98 kg; Table 1). Phenotypically, A. betsileo differs significantly from other eastern woolly lemurs with a pelage that is primarily light reddish-brown on the body and on the dorsal surface of the extremities (Figs. 23-24). A. betsileo has a distinct facial mask with grayish pelage under the mandible and the ventral surface of the extremities. A. betsileo has diffuse cream colored eyebrow markings and a thicker pelage on the head which gives a more round or oval-like appearance of the head that distinguishes it from the other eastern woolly lemurs. The venter is dark gray towards the midline and diffuses to a light gray ventrolaterally. The tail is primarily reddish brown, darker on the dorsal surface than the ventral portion which is a lighter reddish blonde. Diagnosis. —In the D-loop and PAST sequence fragments, A. betsileo differs from the closest rela¬ tives, A. peyrierasi ty pe 2, and A. peyrierasi type 3 by 4.75%±0.9l% (28 informative sites) - 2.92%±0.37% (68 informative sites) and 3.43%±0.77% (19 informa¬ tive sites)- 1.33%±0.23%(31 informative sites ), respec¬ tively. In the D-loop and PAST sequence fragments, A. betsileo differs from the closest species relative to geographic distance, A. laniger, by 5.50%±0.91% (42 informative sites) and 3.45%±0.37% (99 informative sites), respectively. Distribution. — A. betsileo is currently known in the Bemosary Classified forest (Fandriana) and the regional distribution will be tentatively set from south of the Mangoro River and north of the Mananjary River, until its distribution can be confirmed. Comparisons and Remarks.—Avahi betsileo is approximately the same size as A. peyrierasi type #2 and A. peyrierasi type #3, larger than A. raman¬ antsoavana , and smaller than A. laniger (Table 1). Although A. betsileo is not genetically that different from both types of A. peyrierasi based on absolute and percentage differences, the phenotypic differences are noticeable (Figs. 23-24). Additional survey work is required to determine the southern and northern range of A. betsileo and the northern extent of A. peyrierasi type #2 and #3. Etymology. —The name betsileo is proposed for this species and is derived from the Malagasy tribe from the Fandriana region. Vernacular Name. —Betsileo woolly lemur. By applying the phylogeographic studies by Thalmann and Geissmann (2000) and Pastorini et al. (2003), geographic barriers can be defined that affect multiple taxa ultimately of both flora and fauna. By establishing and prioritizing these phylogeographic regions, conservation management decisions can be implemented that will support the preservation of bio¬ diversity within these defined regions. These areas of discrete or unique biodiversity show that the all major rivers are significant factors or barriers and augment the processes of speciation (Fig. 25). The emerging data display a distribution of unique biodiversity Andriantompohavana et al.— Woolly Lemurs of Madagascar 45 Mahajamba Mariarano— Belsiboka- Mangoky Mabavavy Noad- Antafondro* Sambi ratio- Irudo Loky 0_J00 km Maevaraa® Sofia Mahavavy Sud Mamngoza Manambalio Manana bole Tsingy dc Bemaraha Tsiribihina Morondavu Fihcrcnana Onilahy tJeifiarivO Anjanaharibe-Sud Antainambalana Mananara-Nord Masoala Maningory Onibc Mantadia Maromizaha Mangoro Fandriana Namorona Ranomafana Lima Faraony Manampatntna Manombo Manama ra Andohahda (Manangotry) Mandmrc ^ Avaki betsifeo Avaki peyrieraxi Type ^2 • ^ Avakipeyrierasi Type ^ Avahi ramanantsativana (P Avahi meridionalis ■ | Avaki ianlger Avaki uirii otar A vahi cieesgi Avaki tii t itfcrtfaib Figure 25. Distribution map of the woolly lemurs of Madagascar - Revised distribution of the woolly lemurs. The letter “A” represents the location of Ankarana Special Reserve. The letter “B” represents the location of Kalambatritra Special Reserve. 46 Special Publications, Museum of Texas Tech University framing the topology between all rivers (Andrian- tompohavana et al. 2006; Louis et al. 2006a, 2006b; Rioux Paquette et al. 2006). This relationship be¬ tween the rivers and biodiversity transcends taxa and is quite remarkable and consistent (Louis et al. 2006b). As discrete species from different genera are being described, their individual distributions can be overlaid, resulting in river bound “pockets” of unique biodiversity (Cover Figure; unique woolly and sportive lemurs forming paired units of distinct biodi¬ versity). The persistent and rapid loss of habitat and the resulting fragmentation of panmictic populations have compelled wildlife and conservation agencies to take protective action according to existing guidelines and information with the ultimate goal of prioritiz¬ ing species and/or sites. The explosive rate of the deforestation in Madagascar, however, has eliminated many of the available options (Green and Sussman 1990). Because haplotypes can be unique to each population, simply preserving one population will not necessarily maintain species-wide genetic variability (Pope 1996; Louis et al. 2006b). Despite the recent revisions of the woolly lemurs, isolated fragments or forests that represent unique habitat in Madagascar, such as Kalambatritra and Ankarana Special Reserves and Masoala National Park, could potentially contain particular or distinctive Avahi species, so additional comprehensive field work and laboratory work needs to be completed (Cover Figure). Acknowledgments This manuscript was supported in part by a grant from the Committee for Research and Exploration of the National Geographic Society (6613.99), Primate Action Grant, and Margot Marsh Foundation Grant. This project would not have been possible without the support of the staff, guides, and drivers of the Institute for Conservation of Tropical Environments, Mada¬ gascar (1CTE-M1CET), as well as the Association Nationale pour la Gestion des Aires Protegees (AN- GAP), Parc Botanique etZoologique de Tsimbazaza, U. S. Fish & Wildlife, University of Antananarivo’s Anthropology Department, and the Ministere des Eaux et Forets of Madagascar. We acknowledge the generosity of Bill and Berniece Grewcock for their long-term support and commitment, which gave the CCR its direction and identity. Furthermore, we would like to acknowledge that this research would not be possible without the incredible support by the Ahmanson Foundation, the Theodore F. and Claire M. Hubbard Family Foundation, and the James Family. We would also like to acknowledge the computer specialists, Patrick Lill, Dana Gilbertson, and Ron Kipple, for creating the web page and documents. Literature Cited Amato, G., J Gatesy, and P. Brazaitis. 1998. PCR assays of variable nucleotide sites for identifi¬ cation of conservation units: an example from Caiman. Pp. 177-190 in Molecular approaches to ecology and evolution (R. DeSalle and B. Schierwater, eds ). Birkhauser Verlag, Basel, Switzerland. Andriaholinirina, N., J.-L. Fausser, C Roos, C. Raba- rivola, I Ravarimanana, D. Zinner, U. Thal- mann, J. U. Ganzhorn, B. Meier, R. Hilgartner, L. Walter, A. Zaramody, C. Langer, T. Hahn, E. Zimmermann, U, Radespiel, M. Craul, J. Tomiuk, I. Tattersall., and Y. Rumpler. 2006. Molecular phylogeny and taxonomic revision of the sportive lemurs ( Lepilemur, Primates). BioMed Central Evolutionary 6:1-13. Andriantompohavana, R , J. R. Zaonarivelo, S. E. Eng- berg, R. Randriamampionona, S. M. McGuire, G. D. Shore, R. Rakotonomenjanahary, R. A. Brenneman, and E. E. Louis, Jr. 2006. The mouse lemurs of northwestern Madagascar with a description of a new species at Lokobe Special Reserve. Occasional Papers of the Museum, Texas Tech University 259:1-23. Arevalo, E., S. K., Davis, and F W. Sites. 1994. Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromo¬ some races of Sce/oropus grammicus complex (Phrynosomatidae) in central Mexico. Systematic Biology 43:387- 418. Andriantompohavana et al.— Woolly Lemurs of Madagascar 47 Baker, C. S., A. Perry, J. L. Bannister, M. T. Weinrich. R. B. Ab- emethy, J. Calambokidis, R. H. Lien, J. U. Lambersen, O. Ramirez. P Vasquez. J. Clapham, A. Ailing, S. J. O'Brien, and S. R. Palumbi. 1993. Abundant mitochondrial DNA variation and world-wide population structure in hump¬ back whales. Proceedings of the National Academy of Science 90:8239-8243. Bandelt, H.-J , P. Forster, and A. Rohl. 1999. Median-joining networks for inferring intraspecific phylogenies. Mo¬ lecular Biology and Evolution 16:37-48. Barrowclough, G F,, and N. R Flesness. 1996. Species, subspe¬ cies, and races: the problem of units of management in conservation. Pp. 247-254 in Wild mammals in captivity (G. G. Kleiman. M. Allen, and FI. Harris, eds.). University of Chicago Press, Chicago, Illinois. Cracraft, J. 1983. Species concepts and speciation analysis. Pp. 159-187 in Current ornithology, Vol. 1 (R. F. Johnston, ed.). Plenum Press, New York, New York. Davis, J. I., and K. C. Nixon. 1992. Populations, genetic variation, and the delimitation of phylogenetic species. Systematic Biology 41:421-435. Ezard, T. H. G., and J. M. J. Travis. 2006. The impact of habitat loss and fragmentation on genetic drift and fixation time. Oikos 114:367-375. Forster, P, A. Torroni, C. Renfrew, and A. Rohl. 2001. Phy¬ logenetic star contraction applied to Asian and Papuan mtDNA evolution. Molecular Biology and Evolution 18:1864-1881. Gonzales, S., J. E. Maldonado, and J. A. Leonard. 1998. Con¬ servation genetics of theendangered pampas deer {Ozoto- ceros bezoarticus), Molecular Ecology 7:47-57. Green, G. M., and R. W. Sussman. 1990. Deforestation history of the eastern rain forests of Madagascar from satellite images. Science 248:212-215. Groves, C. P. 2000. The genus Cheirogaleus: unrecognized biodiversity in dwarf lemurs. International Journal of Pnmatology 21:943-961. Groves, C. P. 2001. Primate taxonomy. Smithsonian Institu¬ tion, Washington, D.C. Huelsenbeck, J. P, and F. Ronquist. 2001. MRBAYES: Bayes¬ ian inference of phylogeny. Bio informatics 17:754-755. IUCN. 2004. IUCN Red List OF Threatened Species: www. lucnredlist.oru. Downloaded on 12 March 2006. Jenkins, P. D. 1987. Catalogue of primates in the British Museum (Natural 11 1 story) and elsewhere in the British Isles. Part IV: suborder Strepsirrhii, including the sub¬ fossil Madagascan lemurs and Family Tarsiidae. British Museum (Natural History), London, England. Jernvall, J., and P. C Wright. 1998 Diversity components of impending primate extinctions. Proceedings of the National Academy of Science 95:11279-11283. Kappeler, P. M., R. M. Rasoloarison, L. Razafimanantosoa, L. Walter, andC. Roos. 2005. Morphology; behaviour and molecular evolution of giant mouse lemurs ( Mirza spp.) Gray, 1870, with description of a new species. Primate Report 71:3-26. Kirkman, T. W. 1996 Statistics to LJse. http://www.physics. csbsiu.edu/stats/. (8 May 2007). Kumar, S., K. Tamura, and M. Nei. 1993. MEGA Molecular Evolutionary Genetics Analysis, Version, 2.1, Pennsyl¬ vania State University. University Park Press, Baltimore, Maryland. Longmire, J. L., G. F. Gee, C. L. Hardekoff, and G. A. Mark. 1992. Establishing paternity in w hooping cranes (Grus americana ) by DNA analysis The Auk 109:522-529. Louis, E. E. Jr., M. S. Coles, R. Andriantompohavana, J A. Sommer, S. E Engberg, J R. Zaonarivelo M I Mayor, and R. A. Brenneman. 2006a. Revision of the mouse lemurs (Primates, Microcebits) of eastern Madagascar. International Journal of Primatology 27:347-389. Louis, E. E., Jr , S. E. Engberg, R. Lei, H. Geng, J. A. Sommer, R Randriamampionona, J. C Randriamanana, J. R. Za¬ onarivelo, R. Andriantompohavana, G. Randria, Prosper, B. Ramaromilanto, G. Rakotoarisoa, A. Rooney, and R. A. Brenneman. 2006b Molecular and morphological analyses of the sportive lemurs (Family Megaladapidae: Genus Lepilemur) reveals 11 previously unrecognized species. Special Publications of the Museum, Texas Tech University 49:1-47. Maddison, W. P, and D R. Maddison. 1992. MacClade: analysis of phylogeny and character evolution. Sinauer Associates, Sunderland, Massachusetts. Martin, R. D. 2000. Origins, diversity, and relationships of lemurs. International Journal of Primatology 21:1021- 1049. Mayor, M I , J. A. Sommer, M. L Houck, J. R. Zaonarivelo, P. C Wright, C. Ingram, S. R. Engel, and E. E. Louis, Jr. 2004. Specific status of Propithecus spp. International Journal of Primatology 25:875-900. Milne-Edwards,A., and A. Grandidier. 1875a. Histoire naturelle des mammiferes, Tome I, Texte I. In Histoire physique, Naturelle et Politique de Madagascar, Vol. 6, Les Indrisine (A. Grandidier, ed ). Histoire naturelles des Mammiferes. Imprimerie Nationale de Paris. Milne-Edwards, A., and A. Grandidier. 1875b. Histoire Na¬ turelle des mammiferes. Tome IV, Atlas I. In Histoire physique, Naturelle et Politique de Madagascar, Vol 9 (A. Grandidier, ed. ). Histoire naturelles des Mammiferes. Imprimerie Nationale de Paris. Mittermeier, R A., W. R. Konstant, F. Hawkins, E. E, Louis, O. Langrand, J. Ratsimbazafy, R. Rasoloarison, J. U. Ganshorn, S. Rajaobelina, I. Tattersall, and D. M. Mey¬ ers. 2006. Woolly lemurs. Pp. 237-340 in Lemurs of Madagascar, 2nd Edition. Conservation International Tropical Field Guide Series, Washington, D. C. 48 Special Publications, Museum of Texas Tech University Myers, N. 2000. Biodiversity hotspots for conservation priorities. Nature 403:853-858. Nylander, J. A. A. 2004. MrModeltest v2. Program dis¬ tributed by the author. Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden. Pastorini, J., M. R. J. Forstner, and R. D. Martin. 2000. Relationships among brown lemurs (Eulemurfu/vus) based on mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 16:418-429. Pastorini, J., R. D. Martin, P. Ehresmann, E. Zimmermann, and M. R. J. Forstner 2001 Molecular phylogeny of the lemur family Cheirogaleidae (primates) based on mitochondrial DNA sequences. Molecular Phyloge¬ netics and Evolution 19:45-56 Pastorini, J., U. Thalmann, and R D. Martin. 2003. A mo¬ lecular approach to comparative phylogeography of extant Malagasy lemurs. Proceedings of the National Academy of Science 100:5879-5884. Pope, T. R. 1996. Socioecology, population fragmentation, and patterns of genetic loss in endangered primates. Pp. 119-159 in Conservation genetics, case histories from nature (J. C. Avise and J. L. Hamrick, eds.). Chapman and Hall, New York, New York. Posada, D., and K.A. Crandall. 1998. Modeltest: test¬ ing the model of DNA substitution. Bio informatics 14:817-818. Rasoloarison, R M., S. M. Goodman, and J. U. Ganzhorn. 2000. Taxonomic revision of mouse lemurs (Microce- bus) in the western portions of Madagascar Interna¬ tional Journal of Primatology 21:963-1019. Reed, D. H 2004. Extinction risk in fragmented habitats. The Zoological Society of London 7:181-191. Rioux Paquette, S., S. M. Behncke, S. H. O’Brien, R. A. Brenneman, E. E. Louis, Jr., and F.-J. Lapointe. 2006. Riverbeds demarcate distinct conservation units of the radiated tortoise (Geochelone radiata) in southern Madagascar Conservation Genetics. (DOI 10.1007/ si 059.2006.92275.x). Ronquist, F., and J P. Huelsenbeck. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574. Rumpler, Y., S. Warter, C. Rabarivola, J.-J. Petter, and B. Dutrillaux. 1990 Chromosomal evolution in Mala¬ gasy lemurs XII. Chromosomal banding study of Avahi laniger occidentalis (syn Lichanotus laniger occidental is) and cytogenetic data in favour of its classification in a species aparKTiz//?/ occidentalis. American Journal of Primatology 21:307:316. Sambrook, J., E. F. Fritch, and T. Maniatus. 1989. Molecu¬ lar cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Press, New York, New York. Saitou, N., and M. Nei. 1987. The neighbour-joining meth¬ od: a new method for constructing phylogenetic trees. Molecular Biology and Evolution 4:406-425. Schneider, S., D. Roessli, and L. Excofier. 2000. Arlequin: a software for population genetics data analysis. Version 2.000. Genetics and Biometry Laboratory, Department of Anthropology, University of Geneva, Geneva, Switzerland. Smith, R. J., and W. L. Jungers. 1997. Body mass in com¬ parative primatology. Journal of Human Evolution 32:523-559. Swofford, D. L. 2001. PAUP*. Phylogenetic Analysis Us¬ ing Parsimony (*and Other Methods). Version 4.0b5. Sinauer Associates, Sunderland, Massachusetts. Telenius, H., N. P Carter, C. E. Bebb, M. Nordenskjob, A. J. Ponder, and A Tunnacliffe 1992 Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718-725. * Thalmann, U., and T. Geissmann. 2000. Distribution and geographic variation in the western woolly lemur (Avahi occidentalis) with description of a new species (A. unicolor). International Journal of Primatology 21:915-941. Thalmann, U., and T. Geissmann. 2005. New species of woolly lemur Avahi (Primates: Lemuriformes) in Bemaraha (central western Madagascar). American Journal of Primatology 67:371-376. Thalmann, U., and T. Geissmann 2006. Conservation assessment of the recently described John Cleese’s woolly lemur, Avahi cleesei (Lemuriformes, Indridae). Primate Conservation 21:45-49. Thompson, J. D. ; T. J. Gibson, F Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX-Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Research 25:4876-4882. Wheeler, Q. D., and N. 1. Platnick. 2000. The phylogenetic species concept ( sensu Wheeler and Platnick). Pp. 55-69 in Species concepts and phylogenetic theory: a debate (Q. D. Wheeler and R. Meier, eds ). Columbia University Press, New York. New York. Wyner, Y. M., G. Amato, and R. DeSalle. 1999. Captive breeding, reintroduction, and the conservation genetics of black and white ruffed lemurs, Varecia variegata variegata. Molecular Ecology 8:S107-S115. Zaramody, A., J.-L. Fausser, C. Roos, D. Zinner, N. And- riaholinirina, C. Rabarivola, 1. Norscia, I. Tattersall, and Y. Rumpler. 2006. Molecular phylogeny and taxonomic revision of the eastern woolly lemur ( Avahi laniger). Primate Report 74:9-22. Andriantompohavana et al.— Woolly Lemurs of Madagascar 49 Zhang, D.-X., and G. M. Hewitt. 1996. Nuclear integrations: challenges for mitochondrial DNA markers. Trends in Ecology and Evolution 11:247-251. Addresses of authors: Rambemntsoa Andriantompohavana University of Antananarivo BP 906 Antananarivo 101, Madagascar E-mail: radarn byHDZ@yahoo.fr Runhua Lei Center for Conservation and Research Henry Doorly Zoo 3701 S. 10 ,h St. Omaha, NE 68107, USA E-mail: leir@omahazoo. com John R. Zaonarivelo University of Antananarivo BP 906 Antananarivo 101, Madagascar E-mail: zaonarivelo@yahoo.fr Shannon E. Engberg Center for Conservation and Research Henry Doorly Zoo 3701 S. HP St. Omaha, NE 68107, USA E-mail: genetics@omahazoo. com Gerard Nalanirina Center for Conservation and Research Henry Doorly Zoo 3701 S. HP St. Omaha, NE 68107, USA E-mail: smallvaovao@yahoo. com Zimmermann, E., S. Cepok, M. Rakotoarison, V. Zietemann, and U. Radespiel. 1998. Sympatric mouse lemurs in northwest Madagascar: a new rufous mouse lemur spe¬ cies (.Microcebus ravelobensis). Folia Primatologica 69:106-114. Susie M. McGuire Center for Conservation and Research Henry Doorly Zoo 3701 S. 10 th St. Omaha, NE 68107, USA E-mail: genetics@omahazoo. com Gary D. Shore Center for Conservation and Research Henry Doorly Zoo 3701 S. 10 lh St. Omaha, NE 68107, USA E-mail: genetics@omahazoo. com Justin Andrianasolo Center for Conservation and Research Henry Doorly Zoo 3701 S. HP Si Omaha, NE 68107, USA E-mail: srna/lvaovao@yahoo. com Kelly Herrington Center for Conservation and Research Henry Doorly Zoo 370I S. 10 ,h St. Omaha, NE 68107, USA E-mail: genetics@omahazoo. com Rick A. Brenneman Center for Conservation and Research Henry Doorly Zoo 3701 S. 1(P St Omaha, NE 68107, USA E-mail: rabr@omahazoo. com 50 Special Publications, Museum of Texas Tech University Addresses of authors (cont.): Edward E. Louis, Jr. Center for Conservation and Research Henry Doorly Zoo 3701 S. 10 th St. Omaha, NE 68107, USA E-mail: edlo@omahazoo. com E-mail: (Lab - United States): genetic s@omahazoo. com Email: (Field - Madagascar): kelynewsl@yahoo. com Andriantompohavana et al.— Woolly Lemurs of Madagascar 51 Appendix I The following Appendices to this publication are available online at the indicated website addresses. a. Avahi Field Data Appendix (Individual data hie for each Avahi, 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/Avahi.htm b. Appendix Primer Table I (Summary of designed primers for D-loop and PAST fragments). http: //www. omahazoo. com/ccr/genetic s/papers/appendixAvahiprimertablel. pdf c. Appendix Haplotype Table I (Summary of designated haplotypes for Avahi from Fandriana and Ranomafana localities for D-loop and PAST fragments). http: //www. omahazoo, com/ccr/genetic s/papers/Avahihaplotvpetablel .pdf d. Appendix ANOVA dendrogram of the morphological data summarized in Table 1. http ://www. omahazoo.com/ccr/genetics/papers/appendixAvahimorphodata. pdf Sample (98 Avahi and 25 outgroups total) used in the present genetic study and taxonomic revision of the Madagascar lemur genus Avahi, "TK Number is re ferenced voucher curated at the Museum of Texas Tech University. h Mitochondrial DNA sequence data for D-Loop (IN Loop or control region) and PAST (Pastorinifragment) for each sample are available from Gen Bank under the listed accession number . N/A signifies that sequence fragment is not available . (A. betsileo was formerly referred to as Avahi species nova 52 Special Publications, Museum oe Texas Tech University oc ri O' rn -pj- in VO oo O' o CN in o in in a > a a O' CX 2 £ lL Pl pL, o < D < Q a 2 Q UJ UJ UJ w Lj m m vO vO r- oo O' Tj- in vO t"v oo “p, T" o' r- O' r^ in r-' r- in yfi m m Q CP' X T O' r- O' Ov sO vO ■o vO vD m X >n -rt m in in 1 a m oc a 6C in >- m oe O' OC »n > m &o a oo tZ 'n 30 a in oo 2 O' X u. O' X u. O' X Li. O'. X a < < n\ UQ a Q LLt UJ PU UJ UJ CJ 3J Q. CL Cl CL CL CL Cl CL CL Cl CL Cl CL in >■. >. >, +=l >t >> =M* ;>. >. £* 2' r >s_ ■ '*** **** ■*s« ■»« ■■Si zn !>*■ trj Vj tx 4) 33 'si a 3 C3 tj 33 33 3J 33 V C k A. V. Is. L is. L. L. is i. L. L. _2j ■E _0j ■Si .gi .gj »Sii r ^t ■£i x Qh c L. ji> ^s £ l! 2 l! ITj ■- c = su 5? QJ* Ej” 1 rS C 1 cy cl Cl cl Cu Cl Cl, Cl Cl, ■i> cr. •**» * r, »* ' 1 -■»*. ■ B« ' B-U 1 Si -c -ir ■5: X •X s_ cr. G CJ ? r— 33 > J3 > ts > ij s- !3 > a 33 33 > 33 S' 3S > O Q X X X X X X X X X X X X o L L, L, «s. 6, L v. L. L. r- d 5 ij- 5l) CD cy Hi U -Sf •Sf> .tJj ■Sf .gc r go .go ,gO ■£p 3k> -Ep ■SP s: & £ e: g* p*~ £ •‘fc. E: t~- •*«. S3 £ « 1 (3 3 v-J _5 33 33 33 33 .= eb .2P « ■im* -■* *»» 1 "S* ‘•s* ‘St **s* -^1 -4; -s; -e: -c -S3 -S3 -S3 X £3 a es C3 33 33 33 3i — > > 61 > > > C Q X X X X X X X X >, £ p E E E a Q Q Q Q 2—\ 2“S >, >. Sn >\ _> k >■> r >fc jz n -E JS ■ U! eg JC J3 o eg ■U ns OJ o CO LL 1 C3 o eg <1> s O eg c 63 o 63 o 4— 1 eg c eg >*— » s rs 4—* -2 H — 1 c3 w i2 eg x> ^5 _c -O eg eg eg C3 c3 eg eg £ p ►=s E £ E H A E— H f—' H < < < < < / ^^ '—* eg eg 63 (0 ns eg eg eg eg (0 ?3 di c G E c a G C— e C c a C ,cd ,nj . eg f Cv r ro ,eg ,cg ?g eg 63 nj f3 r* 4-. 4-. U~* 4—t Vi r, ^ l£J i*d eg eg nj eg e5 eg eg rs O' CQ O'. 3 o CO o .—i tN o 1) o 01 5h? o 0) o o o o. C. Cl Cl n £b Cl Cl Cl Cl CL CL CL o. >> >-> 4mmi >-* >a -t—■ >x >> b b n ■J—' b >, 4 —t >, >, ■u £ ' •*-«i ‘ ^ ‘ Tv. •is. • pm ■PM ‘■»a ■hN tt tt if) Lp*. C^j L-. r <> e: tt o ?! t: P C tJ tt tt ■ p 1 L «% “H v. L, Ls. L. t. v. Jp. HI ii -S! S o c *-^* ‘ ^ (3 CL D C/> ■ — S. r\ p s«. 2. S v L. p~S ir^-L i: C tj' sii 5j’ 'y ■ii sa oy. Hi ■ ■ jC r^. Cl Ci, Cl Cl =L tt dj 1 OiJ " “V **»+ ■ *^-. - ^ ■I'm • '-V* • .-m - •m. 1 ->-u ■■PM • m 1 *-m m _^- -5- -c: -s: 'i: "S: -tt -tt fc V2 ■'W ■^3 r™ tt tt “ > c > > > > > u O Q ■^r X X 'tt H z 0 U 1 id ■n "o 1*. i*. ifc. L. “V, L L. V L. K. lw ly tt 01 'Ll Zn n> ta tt ?u til *u 5 -S? ■ g3 ?ic -Sf to .tc ■$£ .tp .£c .a? -SP .Sp sr- mm ■y> .i; cr T-< r-p s; E3 e: s; tt tt Z n-p Pd s 5 ■w !3 p-P. tj tt tt r_> C3 r ^-« '''-w ■^ f— i— . _ _ - r m _ m , m _ U ' ■ -_ . ^ .__ , . r _^ r r Id Of] Ofl ■ — -Zz 'S: -s: ■c: £ •E -5 •tt tt a 5J ts t! tt tt tt L*. ?* > > > > > > > > > > o c > ■^7 y ^r; X ___ >, >. E a" c E Q b Q Q ^ > \ >-i >! ^—>, ,—, ,_, ^^ r-* _g; i" C3 c« sj (3 X S3 S3 «3 j ■ ! f !—'. frt L. h i_ L- 1— Lh )-, i_ 1_ W W z: C3 cd (3 f3 C3 03 S3 S3 C o Ci G UnA '_. L. i_ i_ L, •— — 1— •*** +—* HH 1 4—« C3 C3 S3 S3 C3 03 03 C3 7S t’d C3 _0- .E 1 ,&* E E C. E Cl pE ,E -C -C r 4 -S iT^ r" LiC .— f-; IS IE IE IE JC sz J -1 G >-i t —* o o o o o o o O c o < < < < > > > > > > > > > > '—' ** 'y-' .— (3 C3 r3 CS cd Pd efl 03 Pd 03 03 .— G C p—. CI f—a #—• p— 1 C c: c c c C ,E r i^j cE ,t3 ,nJ ,s ,3 ,c3 r pd ,s3 03 3 Q Q r -f- G_ L—i L- C: U3 UT c3 CZi c3 c3 & C3 pd rd n pd nj rz 7Z _l_> rnlrnJ c i <- n E .— P 3= E s Cl *—i p7-* p— B E E E E B > > > > > > T 3" n WiJ C _D O 5 C C O O C o O o o (N C-J rn *3 £ Z z Z z z z z z Z z z Cl Ch c. vO O' 3 < < < < < < < < < < < o O C 30 30 u cC PC Oi PC ad Ci ci ci PC pc > > > z Catalogue TK Location Original Species Current Species n- L oop b PAST 11 Number_ Number __ Desig nation_ _ Designation ___ _ Ml 1 07 125755 Manombo Avahi lamger Avahi m. ramanantsoavana DQ855985 DQ856062 54 Special Publications, Museum of Texas Tech University rn Tf in c* QO O' o w— cs rn vC C* cc vo vo vo ■o ■o vo VC c- c* c* 1^ r- c* C* c- O o o O o o o O o o o o CM Cl Cl VO VD sC vO vs VC 'O vC vC -c X vO VC VO VO m. m, in ■n m m in m m in in in o' On O' esc oo esc cc cc oc oc oc oc co so so ro m n O a O a o a a a a O' O' o X X X c a n, a c c C o a c a a W ■X X vO r- esc O' O i— ci rn m vD r- 0^ o ,_ cc oc sc esc O' O' O' O' O' ON ON r- cc zr. o O' O' O' o Os O' Ov O'. Ov O' O' (N Cl cl in m 'n m in in in m *n m m m SO vO vo m in in m »n in m in m m m ■n OS ON ON so so oc oc oc oc oo oo cc co cc cc m m o o o a o a a a a a a a Ui X X Q nv c Q D n> ■—■ a c Q a G D U Ll) LU 3 3 13 3 3 3 3 3 r~ r* “V si ’■Sal 3 a 3 3 a 3 S' e* 3 ■sj N ■Nj 3 o 0 ) Q Q o o O i3 *3 3 3 vrs r-- c— “V- c- 3 3 z 5 3 *»» NJ 5 3 <3 r-*- 3 3 s: si 3 P>» —7 <3 —s 3 g 5 13 o o Cj o o 3 H) 3 o HI 3 5 5 5S § " ■ *“H 33 ‘*s* "^s i* K i2 ti; trj i3 £ S 5 lia ~Cs ■Oi He ■Ci Hi *43 ■o HJ ■c Hi *3 ■ ***. ■ «^u. . ^ - ‘■‘’Hi ‘■^i *l^Sr ■»» ri *«- r +H. **Vi ^c- -£ -£ •ec *-c ji *5: 3 5s 3 gs 3 3 ts 3 3 3 3 3 > > S' r 1 *- > > c- > > > X X 'T S, +*m ■s •s. V Is Vi i- 3 lv 5J Hi Hi u as as as as as u ^C- •SP be .£? .^p .EP ■S® ■5p ■ *C .be ■£P .iso *£p .£p p«* £ c* “s. c« ■s, £ 3 tr e~ 3 V*rn •y- •r- £ 3 3 3 3 53 5i 53 3 55 3 53 3 3 3 3 rL 'Sl -^i . r • r_„ ■ ^ " ■*# ' Si - 6 f»i. ‘ *»+ C *^1 ■sE? -3 -3 -3 -SI -3 -3 *3 -3 3 3 3 3 3 3 3 3 3 5E 3 3 3 3 S* r“ > r- c- > s» > > > X X X ~x O c o o o o o o o o to o o C 3 rs J 0 j _n JO JD JD X) X n c c e e c a C B s B E r“ c .2 2 -2 cc ■ .o .2 ,2 o S o l“ cd cs a" o e o c o Q O o o e ns ' ^ T 3 C "U T 3 C n TO c: |m C ’C T 3 C "C T 3 c rj 1 1_ TT O S % 5 S s o X O Lla (0 Ll. K 5 LLi O X m U- 5 (X O SX so c- cc O' m m m m O t- r- c m m m m Cl Ci cs Cl cc o -it m ON o i— I ■—. * —i cs Cl vo c- O'. oc o ON o >n m »n G". •n ■O 1 < '3 ca < ^-=- cs ci ci Z ci Z ci Z vd Z vd Z X Z s 1 < < < < < < < < 2 U- EX X X X X X Appendix II (cont,) Andriantompohavana et al.— Woolly Lemurs of Madagascar 55 wo wo VC r- so O' 0 rJ cO Tf I/O vp O' oc ■*1 r~- r- t" r- T' r-' r- QO «J so oc CO sc CO co =c j— T'l fN o o o O o O 0 O 0 0 0 0 0 CP L or vC so VC VC vO vO VC vO VC O VD 'O vo vo VO vo t>- CT- VO I/O wo wo wo wo WO WO wo wo wo WO wo < rO, rn oc oc SO so oo so so so SO 00 so oc CO cc Q. u. Ui (J- a a a a a O a 0 a a a C/ a Pl: c ro D Q Q Q D Q a 0 p c O 0 cl m cc O' o . CJ co wo VO ro oc CV 0 _D (■v oc oo O' O' o o o O O O 0 0 0 O ,—i /-s (N cn O' O' o o o O O O 0 co 0 O 0 0 w O vo SO WO I/O vO vc vO ■d ■O 0 vO vO vo VO vO VO T, cs 0 s wo wo wo W: 'Cl vo. vo wo wo wo vo wo vo WO ro oc oc 30 SC so SO sc sc 00 oc so 00 > l—i >n 4— 1 2/ -- 2? X 2:‘ X iZ' X 2^ X X Z‘ X X So 4-* X Hi Jxj •3J 'll 5 01 CJ su Hi HI Hi Hi CJ H) y — Cl. o .gp ■‘S+ ■S? .tp .bp .bp ,bp .bp .bp .bp .bp' • SP -SP oo w > > P* IV x ■F-* > ?! > L. c SL L L u p ■^p! T; X or X ■'b T ob •jj _ _ tu Oi :b Cu cu O Hi Hi Hi HI Hi Hi Hi ■Sf .£C .go .60 .bp .bp .bp .bp ,£p .bp .Sf .bp -tp -Sf -SP 00 . ~ $b L*». JSi T*- £ Sb Sb k: 5b & S3 kj 2 bi b3 Cl & •S» fSp sj -5 J3 ~s b; bi bi bs .5 oj> •— 1 . ^ ‘ ■-M. *•»* ■ Tn ■ *« -s: -v: sc -x: sz sz hs; -IT -t ~Sb bs ■X o: Cj CJ bJ b! bj bs bj vb bi J—i CJ 1 > C. > > s* > X X. > > L L > O Q 'T t; 0 : X ™ (JL P, _L U O U O c_ a. 73 ns nl m r- 73 1" ra ■crJ C3 ,E .3 .2 Z .2 Z .2 JC N — « N ■s tx- ■i St a 5 .2 .rtS T3 T3 'tj 00 T3 ■5 ■5 mj 3 s E E E 1 u. ‘ u_ 03 O C3 ej 71 Oi n3 O'. O' O'- O' ‘O' O' O' O' o o O o o o o o o o o o o O' o o —i ~—i — ,- & vO vO vO o n m r m in m m n m in in m r m m in m m ■n in co oc 30 30 30 oO co CO oc oc oc oo oc 30 oo oo Q a a a C 7 a o a a a CX a O' a a a Q c a Q a c a a a Q Q a Q a a Q r *T, r^* _Ln .n . ^ .Jo C>] *«3 ■ *« ■•h CN m nj rN CN CN \s "3 ■•-J C 3 vj \J ■tt =tL Nt St 4 t s 3 : s: r-- C^“ C 3 a> CJ 1 > CJ 0 > ■XJ o o 3 o C! 3 o O CL CL CL CL CX tx en a> “!*» % Cx 4 -^ >■> w >v £> 3 ' §>h X X X X 2 ! n% ■X X ■> X X u e cx o co Xi X) s> Xi XJ ss ■x. £ .^p ■Sf .bp - 5 £ V> ■- £ £ sr A. e: •»»* X •^v Ej si K si 3 cs es a C 3 C 3 c g “*■ " V *N» a> cjj +—i ■ ■l». r —^ 1 1 •** “Hh» -x* ’•x. -s: ^7 -or _rx Nr nt NT Nr NT NT Lm t/Z J: 5 ! Ss vi Cj vi -3 t 3 NJ vl r: C3 C3 rs r-* 3 S» > X > > X O Q N- X c z tin a. a. < O o '■-V. X X L-. X X X X X 3 x„ X CJ ,_ 50 5 J qy X to Lt w ty 5 J r-i. Ei ■ n* [3 ,^C .bp ,00 ■SP ■Sf> £f - 5 p SP ,SP -Sf> . 5 ? (/: .a 11 j ■F' r-. “-T £ Fvi -*Si SXi -x sr sr ■ 7 ; to ■sj b: <3 n n n a 2 = r ^“ -.. .= -i> hfi ■ “ N: -5 -S ‘■» ~sr --n £ r: n 9 b! bs -3 t 3 bj ‘— a> > X X r* x r** L X c 0 X X X n: X ~ b c C Lj O 0 0 0 tO o/> S/j olj P—1 c z . s 03 (3 n-t C r“ F^-- s co rs s > S ■-“N X-> cz J3, C3 (5! aj CJ U CJ r~ r -1 J= rs CS “ I—* n-i JZ 3L O O O O -O T3 T3 1 “ r“ c C < < < < ? ^ cn t eg i 2 O +—' eg c 3 O ^-> oO 3 J 0 SJQ 1 “ r- Z z O C 3 cc eg > 5 S 3 eg El eg *—* S g CC — x—N O Cg -2 1 f 3 CJ D OJ o z A x. Cg e eg c eg to 3 T3 X. O Z i 2 p 00 EiO (fl eg eg -N tg eg eg z 3 0 O O > > > _ H --. T5 ■a Tg X X 0 O 0 ^■, Z z i eg 1 « i-ri S—1 i-. eg rt nj Z j—1 El (g s ?3 c r—* E eg ra eg s s CJ m ■TT ’-T; VO ■O vO VO f— n- r- r^ t- V/ x n n n * fE n CN (N H Z 0 cn u ~ CN — 1 *— — — 00 b —, 01 m 0 C^j tT TT Tf’ Tf Tf Tf’ ^ _E TT m O' ’—« rn m < < < < < < S E c Q Q a a 5 Q c a cd ctZ K C£^ 2 C & b ej z Z Z z z z z Z z z < < < < < < 0 z < < < < < < < < < < Z z z Z z z Catalogue TK . Original Species Current Species b u . tT b vt L u a Location ~ . . r ^ t D-Loop HAS l Number_ Number ___ Designation _ Desig nation _ J_ NARA4.18 125767 Mananara-Nord (Ivontaka-Sud) Avahi laniger Avahi laniger type #2 DQ856028 DQ856105 Andriantompohavana et al.— Woolly Lemurs of Madagascar 57 so t— CO O' o 1—* 22 so SO r- oo o o o o o — ■—* 1 *—* — 5 ■ 2 CX Cl CL £r‘ L. !3 c o _o o * ‘♦i* ■ “»* S3 l "3 "5 vy 'O ft) Xi .Si SJ j .Si Xl ts* **- § £ s £ £ Jlj o Si Si Si SJ a ~G Sj ■o 2j Cs D 31 2b J*- ',F, -S3 ^£ r, h^, -s: -5 ■ -S3 ■ -S3 ro S3 si 3: 53 3 31 31 > > X > £**■ > c> > > Ss > X X X X x X x X ■o -is ■Ea ,Eg ■". 33 "S "5 , Q -2 ■ 1 S3 *Ft K 5 be s 5 .bC o s si xi ■o 5 in XJ Cf, XI jx l> J3 s .Si £ SJ ■ >-■-* ,o T** ***. xi Si XI -2 XJ D 31 31 O X) SJ Si <3 Si 3 31 ’o 23 ■Si 2j 2j O O O ■►v. 'P6* >Si ■ -m*. ■- *■!»* -«» 4 r** -S3 m f^- «^S: -3 -S3 33 J3 -S3 -C -g -J3 31 ^3 31 3! 31 3 3S 31 3i ro 31 33 t3 33 > 3> > 3- > > ;> o > X X X X X X X X X X X "^5 TO 3 3 {/: LO sXJ 5 A: 2 4CJ 2 O > r^; 5 > E E Cd e _c5 £ se ^3 efl J=: is j=, 73 -S cd ■s o o o 0 C3 rfl «3 n, »^-« r ' k- 1-4 1_ u 1-, !_ i_ u Li 'p 5 1 r ; TO c ?3 E3 es ^5 CJ C3 (C n3 m <■-/ i- o g s g S B u E 3 1—. c iy E i> E Ci PT o O D CQ OQ CQ £2 CQ 1 RS if r3 S-i i XS ie- CS u ■—C C L-. o c i_ T3 c L- "O i —<■ m *o a> TO o TO o 4i 2 fO 5fl c cB c oB c >;. oa c _c Li .2 'Li L 2 Li 2 4 L- 3. cd 5 C < c < E < £ < U1 E— cr H * s H t/i F—I trt H cd cd cd 2 cc O' o —i CN CO Tf so r- r- r- O- »■ t> r- t" r- r- vo 50 >o so SO, so OJ rl cn: or OS c-i O'] co —■ OJ CN co CO o or r-] co or Tf of Tf OO Cs oc O' — “■* -—. O' so CN < < vo to SO) < < < < < x fE so ll c£ H h- H u £ 2 £ < < < Z z z Z UJ ll ll: s ue < >■ i < < —H Z z z < < < < ee OQ 09 2C ac S s 58 Special Publications, Museum of Texas Tech University PI n rf VP

P tp oc oc CO co CO CO cc DO OC oc oc < =C oc 50 OC oc 'p >P tp pi ■p ip ip tP tp ip ip O' a a o a > >" >- > > p" > > > > a r*' P, G G < < < < < < < < < < < TT tp VO co *P PI cc o rf oc cp PI o CO cx rr rr rr -rr rr Ov PI (O 'P cv rp Ov Ov to so o o o o vo r-' PT o 'S' p- tT tr rr rfr rr v—<■ vo vO vo vO O O ov TT rr rr ip rr rT vt rr w *p ip > > > > > > > > D Cl c a G < < < < < < < < < < < i ■ eg -j£ ca :z P _> 0 O o o o 3_ £ c eg c 73 S 5 5 r eg Or, CCJ eg G/J . W 1 — t- P3 p rd S eg 2 3 C3 L* C3 o c a o > 4 s- eg E 'C eg s "C fS i—« cd s eg £ j; C < C 53 tti s s K3 p t*- tfj g 53 c .2 5 0 — P3 p— i— eg o ■a C S— O — O s' p-l S c 53 so 53 C3 c 75 G r* S f3 C3 ■ ci Uh Qi < < oi ed JD E Z o £ ■3 £ cc =3 U Z rr ip vO o ,_j tp ip 'P vo vo dc; cd CiL u. < < < < < 2 z < a pv tp PI tp. pi PI rr c oc o pi o Z z z z < < < < < al a c2 llw o=: co PI _ __ PI pi \D vo PI Ov o o ■y < z PC < z < z A < < o 2 Appendix II (cont*) Andriantompohavana et al.— Woolly Lemurs of Madagascar 59 r- o O' o ■0 r- tt %r tn •rt in in n E— 1 OT m m > > a a > < < < i—( Q < < O' o' oo O'- T' O' 0 CN 50 > > a a a a >■ a a a O' U- < < < D Q c a < a n 0 a O < \ ■ V r— • “V Srt. vl V 2 in — j *»+ a ‘•h ■ Hu, rt 5 rt 5 ■a ■■0 ^3 £ V <3 Jfc V o> t § !> ■ilN^ s: -g c y ■V Li **v* C3 £ 0J ■»vj 3! cu t ■S Sv £ k. *vi C3 5! O 'J Hi 8 13 Si > » *v ‘V., ■v* Vrt* ss a a a cr, *-> co c c Sta. 'O ■o U G L> Ll Li ■v ^5 ^2 0> L> L> #»* '-w ! 1 s- £ f =£: *1^ 1 '-M, "‘Hu. L *»v,. *L ■•v, g- £ cu 5 *Tt Cu- 3 g Crt V* S 3 'S -V. 2 V ^5 [rt ■HJ ■ 1 ^ "5 3 mi 'tr ^3 t s 5u O L> * "m, £ CD '•v* C3 J) C V G « c £ C<3 Vu. L_i 53 CJ % 3* SJ 0 u. oj ■’S TS ’*>w 01 S>- to (rt [rt, rt rt rt rt rt CL S 1*. Hu— -V. V* r ^ ..__ ■v4 --_ 51 =3 ■4 Mj a ’■Vi a {/) .a ■Ki X CJ v> U CJ Cj ■Li Cj G L> <>- >3 i. V V o a *—* V, “V rt —-, CX- Oh CL CL CL CL CL : cO 1 : ■ X3 T3 ■0 cO 3 3 (O c n cn 1 E o 0) co (O 0 co CO c3 co C3 X3 (H CQ E3 C c: 0 (rt O c r e0 +— r3 r ^: -+— CZ ,co l+H (0 cd cd b N 1—1 C5 Jj .2 .^3 ■U 0 •a CJ £ C’d cO JxC Q cO CJ c s o c co IT"! o 5 3 «—< CO 3 r H” c p cd cO r-“ cd E 1 "O co H eo E 0 c 73 — ^di 3 03 O 111 c vi (0 X3 03 lO > cO O X3 O V- y £2 Qd SsC < < < CD f— < Q m V oo M n on Vi in rn m 0 n) do G O jz on m m >n c cn m TT o 00 o o c O T ni < T^ ** 10 cO £ 2 2 2 -2 K ci a 2 < pd DC QC rtl to 2 < < < < < < 2 C tIJ < c O Z OcC oC h a < CQ ac Q s