DSpace at VNU: Molecular phylogeny of Nycticebus inferred from mitochondrial genes

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International Journal of Primatology, Vol 27, No 4, August 2006 ( C 2006) DOI: 10.1007/s10764-006-9032-5 Molecular Phylogeny of Nycticebus Inferred from Mitochondrial Genes Jing-Hua Chen,1,2,3 Etsuo Narushima,7 Vu Ngoc Thanh,10 Yun-xin Fu,3,11 and Deng Pan,1,3,4 Colin Groves,5 Ying-Xiang Wang,6 Helena Fitch-Snyder,8 Paul Crow,9 Oliver Ryder,8 Hong-Wei Zhang,2 Ya-ping Zhang 1,3,12 Received January 24, 2005; accepted February 22, 2005; Published Online September 12, 2006 Researchers are still discussing the classification of Nycticebus We established a molecular phylogeny covering all recognized taxa in Nycticebus to provide information for further evaluation We sequenced partial D-loop (ca 390 bp) and cytochrome b genes (425 bp) from 22 specimens We separated most of the major groups except for some mixing of Nycticebus coucang coucang and N bengalensis Nycticebus pygmaeus diverged earlier from the ancestral stock than the other taxa Nycticebus coucang menagensis was well discriminated from N c coucang It may be possible to explain the mixing of Nycticebus coucang coucang and N bengalensis by the hybridization between the groups in the field Although our data did not provide direct Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, 650223, China of Life Science, Shandong University, Jinan, 250000, China Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, China The Graduate School, Chinese Academy of Sciences, Beijing, 100039, China School of Archaeology & Anthropology, Australian National University, Canberra, Australia Department of Phylogensis and Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, 650223, China Ueno Zoological Gardens, Taito-ku, Tokyo, Japan Zoological Society of San Diego, San Diego, California USA Kadoorie Farm & Botanic Garden Corporation, Hong Kong, China 10 Department of Vertebrate Zoology, Vietnam National University, Hanoi, Vietnam 11 Human Genetics Center, University of Texas at Houston, Houston, Texas USA 12 To whom correspondence should be addressed; e-mail: zhangyp@mail.kiz.ac.cn ∗ Jing-Hua Chen and Deng Pan made equal contributions to the work School 1187 0164-0291/06/0800-1187/0 C 2006 Springer Science+Business Media, Inc 1188 Chen et al evidence for or against the new proposal that Nycticebus coucang javanicus be raised to the rank of a distinct species (N javanicus), we have good evidence for regarding N c menagensis as a species KEY WORDS: classification; mitochondrial gene; molecular phylogeny; slow loris INTRODUCTION Slow lorises (Nycticebus spp.) are small and sluggish primates designated as endangered according to IUCN (http://www.redlist.org) The classification of Nycticebus is under debate Speciation in Nycticebus may have commenced some Myr ago (Lu et al., 2001), but the morphological similarity makes it somewhat difficult to delimit species and subspecies, and consequently different classifications are current, the number of species varying from to (Cheng et al., 1993; Corbet and Hill, 1980; Dao, 1960; Ellerman and Morrison-Scott, 1951; Groves, 1971, 2001; Hill, 1953; Ma and Wang, 1988; Petter and Petter-Rousseaux, 1979; Wang et al., 1996; Zhang et al., 1993) The most popular classification is the 2-species version (Nycticebus coucang and N pygmaeus) that Ellerman and Morrison-Scott (1951) first proposed and Groves (1971) definitively established Dao (1960) proposed an intermediate species (Nycticebus intermedius) in Vietnam, but researchers showed it to be the adult form of N pygmaeus (Cheng et al., 1993; Groves, 2001; Wang et al., 1996; Zhang et al., 1993) More recently Groves (2001) maintained that there are species in Nycticebus: N coucang, N pygmaeus, and N bengalensis, and that N coucang has subspecies: Nycticebus coucang coucang, N c menagensis, and N c javanicus, among which the position of the Java subspecies was still in doubt Groves now agrees with Supriatna and Hendras (2000) that the Java form should be set apart as a distinct species, Nycticebus javanicus (Groves and Maryanto, in press), giving species in Nycticebus Previous classifications of Nycticebus relied mainly on morphological differences, including body size, pelage color, head forks, and other characters Because the species and subspecies boundaries in Nycticebus are still unfixed, more taxonomic characters other than morphological ones may be useful for further evaluation So far, however, studies on karyotype (Cheng et al., 1993), restriction enzymes (Zhang et al., 1993), and DNA sequences (Wang et al., 1996) have concentrated only on resolving the relationships among Nycticebus bengalensis, N intermedius, and N pygmaeus Researchers have so far not undertaken molecular studies attempting to reconstruct the whole phylogeny of all the taxa in Nycticebus Phylogeny of Nycticebus 1189 To further the understanding of phylogeny and classification of Nycticebus, we have partial D-loop and cytochrome b (Cytb) genes from all recognized taxa in Nycticebus and established a molecular phylogeny We aimed to test whether the evolutionary relationship of all the taxa in Nycticebus would be concordant with those based on morphology and to provide molecular information for further evaluating the classification of Nycticebus MATERIALS AND METHODS Samples, DNA Extraction, Polymerase Chain Reaction, and Sequencing We collected 22 specimens including Nycticebus pygmaeus and N bengalensis, as well as specimens identified from photos as Nycticebus coucang javanicus (1), and probably N c coucang (3) and N c menagensis (5) (Table I) We extracted total genomic DNA from hair, blood, or tissue We purified hair samples via a hair DNA Purification Kit (Bio-Rad) and extracted blood and tissue samples via a proteinase K phenol–chloroform method with isopropyl alcohol preparation (Sambrook and Russell, 2001) We used primer pair L15996 (5 -CTCCACCATGAGTAGCACCC AAAGC-3 ) and H16498 (5 -CCTGAAGTAGGAACCAGATG-3 ) to amplify the first hypervariable region of the D-loop Polymerase chain reaction (PCR) cycles contained an initial denaturation at 95◦ C for min, and then 35 cycles each consisting of denaturation at 94◦ C for min, annealing at 50◦ C for min, and extending at 72◦ C for min; followed finally by postextension at 72◦ C for 10 We purified amplification reaction products via the DNA Isolation & Purification Kit (Watson) We performed direct sequencing on an ABI377 DNA automatic sequencer We sequenced both strands to confirm sequencing reliability Because Lu et al (2002) reported the presence of a nuclear pseudogene (numt) in slow loris, we also sequenced partial Cytb genes from samples representing the major branches of the D-loop tree (Fig 4) to test whether the phylogeny of Cytb was still congruent with that of D-loop If so, we might be able to reduce the possibility of unexpected numts because mitochondrial genes constitute a single locus and the likelihood that both genes are numts is low Meanwhile, because the samples selected also represent all recognized taxa, we would be able to evaluate the phylogeny and classification of Nycticebus from Cytb as well We used general primer pair L14724 and H15915 (Irwin et al., 1991) to amplify Cytb genes, and other procedures were the same as those for the D-loop 1190 Chen et al Table I Taxa, individual codes, localities, and GenBank accession numbers Accession no Taxa Individual code Localities Nycticebus pygmaeus P1 Hekou, Yunnan, AY875959 China AY875960 AY875961 AY875962 Boundary AY875941 between China and Vietnam AY875942 AY875943 AY875944 AY875945 AY875946 AY875947 AY875948 AY875949 Confiscated in AY875955 Japana Nycticebus bengalensis Nycticebus coucang coucang Nycticebus coucang menagensis Nycticebus coucang javanicus P2 P3 P4 B1 B2 B3 B4 B5 B6 B7 B8 B9 C1 C2 C3 M1 M2 M3 M4 M5 J1 Malaysia (presumably Malaysian Borneo) Javaa D-loop Cytb AY878366 AY878367 AY878368 AY878360 AY878362 AY875956 AY875957 AY875950 AY878363 AY878364 AY878361 AY875951 AY875952 AY875953 AY875954 AY875958 AY878365 a The exact original of these individuals is not known Groves made the identifications before running the DNA analyses Photos of these individuals are available on request to the corresponding author Data Analysis We manually checked DNA sequences for accuracy and aligned them via MegaAlign in DNAStar software We calculated summary statistics such as the number of variable sites via Mega2 version 2.1 (Kumar et al., 2001), as well as the relative rate test (Tajima, 1993) We tested saturation of transversions via DAMBE (Xia, Xie, 2001) using K80 distance (Kimura, 1980) We constructed phylogenetic trees via both maximum likelihood Phylogeny of Nycticebus 1191 Fig Localities of samples Triangles and circles indicate the places of origin of our samples of Nycticebus bengalensis, N pygmaeus, and N coucang menagensis, respectively The places of origin of our samples of Nycticebus coucang javanicus and N c coucang are unknown; we have placed squares and diamonds (respectively) to indicate their approximate distributions (ML) and maximum parsimony (MP) methods with the help of PAUP4.0∗ (Swofford, 1998) with a 1000-step bootstrap We selected the DNA substitution model for the ML tree via Model Test 3.5 (Posada and Crandall, 1998) We extracted outgroups used for the D-loop and Cytb trees from the mitochondrial genome of the ring-tailed lemur (Lemur catta) (GenBank accession no NC 004025) RESULTS Sequence Variation We deposited all sequences in GenBank, and accession numbers are in Table I We obtained ca 390 base pairs (bp) of partial D-loop fragment and 425 bp of partial Cytb fragments We found 17 insertions or deletions 1192 Chen et al Fig Transition and transversion frequencies vs K80 distance (A) D-loop and (B) Cytb, with Lemur catta as outgroup (C) D-loop without any outgroup S denotes transition and V denotes transversion A shows that transitions are saturated while in B and C they are not, which means that it is not appropriate to use all the sites to recover the D-loop tree if Lemur catta is the outgroup Phylogeny of Nycticebus 1193 in D-loop sequence alignment but none in Cytb Apart from gap sites, we scanned out a total of 72 (18.5%) variable sites in D-loop alignment; 60 of them were parsimony-informative For Cytb, 74 (17.4%) sites were polymorphic and 55 informative Most substitutions were transitions: 79.3% in D-loop and 80.5% in Cytb In Cytb, 70.8% of transitions were at the 3rd positions, 3.8% at 2nd positions, and 25.4% at 1st positions Variable sites of the D-loop and Cytb are summarized in Tables II and III, respectively We found no unexpected stop codon while translating Cytb sequences, which obviates one of the main suspicions of being numts We tested transition saturation before constructing phylogenies by plotting transitions and transversion vs divergence; in our study this was at a distance of K80 (Kimura, 1980) For D-loop data, with inclusion of outgroup Lemur catta, transitions showed saturation and, without inclusion, we did not detect any saturation For Cytb, no saturation existed even with inclusion of Lemur catta Phylogeny The hypothesis of a molecular clock was upheld in all the trees we built when including all the sites Model Test for ML trees selected the HKY + G model (Hasegawa et al., 1985; Posada and Crandall, 1998) in both D-loop and Cytb whatever outgroup we used ML and final consensus MP trees showed a similar topology in all cases, so we present only the ML trees here At the same time, we also applied constraints other than the best MP trees and the lengths of the constrained trees are all longer than the best corresponding MP trees At first, we tried to build the D-loop tree (Fig 3) with Lemur catta as outgroup using all sites even though transition saturation had been detected, because we thought it inappropriate to construct the tree with only transversions, which occupied 50) are placed above the corresponding branches The D-loop was well resolved The branch length from lemur to the root of the other taxa in Cytb tree is 1.31939, unlike in Fig 2, which is not too much longer than inner branches For abbreviations see Table I Phylogeny of Nycticebus 1197 1198 Chen et al to our phylogeny, samples of Nycticebus bengalensis form a clade together with specimen of N coucang coucang One explanation might be that, because there is considerable gene flow between the taxa in southern peninsular Thailand (Groves, 2001), it is possible that this particular specimen of Nycticebus coucang coucang may be from the border region; though it is known that our specimens of N bengalensis came from the China/Vietnam border region, we not know the exact area that served as the source of our samples of N coucang coucang; the bootstrap values are not high Nycticebus coucang According to Groves (2001), Nycticebus coucang contains subspecies, of which (N c menagensis and N c coucang) are difficult to distinguish with full confidence by morphological characters, but they are well discriminated in both D-loop and Cytb trees, which suggests that mitochondrial markers might be very effective in discriminating the subspecies It is possible easily to distinguish Nycticebus coucang javanicus from the other subspecies by its creamy neck and sharply marked black head forks Its apparently consistent difference from the other taxa was the reason that Groves and Maryanto (in press) regard it as a distinct species Our single specimen is not, however, strongly differentiated molecularly from Nycticebus coucan coucang, though in the D-loop analysis it does form a sister clade to the N c coucang plus N bengalensis clade The figures for the presence or absence of I2 are interesting to consider here In Nycticebus coucang javanicus and N c menagensis the tooth is always absent, in N c coucang 20% lack it and 80% possess it, and in N bengalensis it is always present (Groves, 2001) In this character, Nycticebus coucang menagensis is closer to N c javanicus than to N c coucang, while N c coucang is closer to N bengalensis Because in our phylogeny, though not fully resolved, the Nycticebus coucang menagensis clade was clearly more distinctive than the other subspecies, and if N c javanicus could be considered a distinct species, we suggest that N c menagensis might also be considered as a species CONCLUSIONS Scarcity of samples and limited field information hinder us from a thorough understanding of Nycticebus spp This is the first study of molecular phylogeny that covers all recognized taxa in Nycticebus Because their taxonomic relationships are still questionable, our study may provide useful Phylogeny of Nycticebus 1199 information pertinent to the classification of the genus The molecular phylogeny we recovered, though not fully resolved, provides an insight into the relationship of all the taxa, which could form the basis for a range of future studies in ecology, evolution, and conservation of Nycticebus Because of the limited samples and genetic markers, however, there is still some uncertainty around the incongruence with morphological studies, particularly the relationship between Nycticebus bengalensis and N coucang coucang and the taxonomic position of N c javanicus and N c menagensis Such questions need more extensive molecular genetic studies, with more samples (of known origin) and markers ACKNOWLEDGMENT This work was supported by grants from national 973 project of China, National Natural Science 243 Foundation of China (30021004, 30430110), Natural Science Foundation of 244 Yunnan, and Chinese Academy of Sciences REFERENCES Cheng, Z P., Zhang, Y P., Shi, L M., Liu, R Q., and Wang, Y X (1993) Studies on the chromosomes of genus Nycticebus Primates 34: 47–53 Corbet, G B., and Hill, J E (1980) A World List of Mammalian Speices, British Museum (Natural History), London Dao, V T (1960) Sur une nouvelle 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understanding of phylogeny and classification of Nycticebus, we have partial D-loop... the topology of the Cytb tree Given that Nycticebus pygmaeus is deeply separated from other taxa on our molecular data, we concur that it is a distinct species Nycticebus bengalensis Nycticebus. .. and circles indicate the places of origin of our samples of Nycticebus bengalensis, N pygmaeus, and N coucang menagensis, respectively The places of origin of our samples of Nycticebus coucang