Alouatta Palliata Mexicana) in the Selva Zoque, Mexico

Primates (2014) 55:155–160 DOI 10.1007/s10329-013-0399-6

NEWS AND PERSPECTIVES

Limited genetic diversity in the critically endangered Mexican howler monkey (Alouatta palliata mexicana) in the Selva Zoque, Mexico

Jacob C. Dunn • Aralisa Shedden-Gonza´lez • Jurgi Cristo´bal-Azkarate • Liliana Corte´s-Ortiz • Ernesto Rodrı´guez-Luna • Leslie A. Knapp

Received: 9 October 2013 / Accepted: 6 November 2013 / Published online: 26 November 2013 Ó Japan Monkey Centre and Springer Japan 2013

Abstract The Mexican howler monkey (Alouatta palli- consistent with a recent and mild population expansion and ata mexicana) is a critically endangered primate, which is genetic diversity appears to be historically low in this paleoendemic to Mexico. However, despite the potential taxon. Future studies should use a combination of mito- signiﬁcance of genetic data for its management and con- chondrial and nuclear markers to fully evaluate genetic servation, there have been no population genetic studies of diversity and to better understand demographic history in this subspecies. To examine genetic diversity in the key A. p. mexicana. These studies should be undertaken remaining forest refuge for A. p. mexicana, the Selva Zo- throughout its geographic range in order to evaluate pop- que, we ampliﬁed full-length mitochondrial control region ulation structure and identify management units for con- sequences (1,100 bp) from 45 individuals and found 7 very servation. Due to the limited distribution and population similar haplotypes. Haplotype diversity (h = 0.486) and size of A. p. mexicana, future conservation strategies may nucleotide diversity (p = 0.0007) were extremely low need to consider genetic management. However, a more compared to other Neotropical primates. Neutrality tests, detailed knowledge of the population genetics of the sub- used to evaluate demographic effects (Tajima’s D = species is urgently recommended to maximise the conser-

-1.48, p = 0.05; Fu’s Fs =-3.33, p = 0.02), and mis- vation impact of these strategies. match distribution (sum of squares deviation = 0.006, p = 0.38; raggedness index = 0.12, p = 0.33) were Keywords Population genetics Genetic diversity Alouatta palliata Primate Bottleneck Conservation J. C. Dunn (&) J. Cristo´bal-Azkarate L. A. Knapp Division of Biological Anthropology, University of Cambridge, Cambridge CB2 3QG, UK Introduction e-mail: [email protected]

J. C. Dunn A. Shedden-Gonza´lez E. Rodrı´guez-Luna The Mexican howler monkey (Alouatta palliata mexicana) Centro de Investigaciones Tropicales, Ex-Hacienda Lucas was originally distributed from southeast Mexico to Martı´n, Xalapa, VER C.P. 91019, Mexico southern Guatemala (Rylands et al. 2006). However, owing A. Shedden-Gonza´lez to widespread habitat loss and fragmentation throughout its School of Applied Sciences, Bournemouth University, Talbot range, numbers have declined drastically over the last Campus, Poole, Dorset BH12 5BB, UK 30 years and this subspecies is now restricted to highly fragmented forested areas in the Mexican states of Vera- L. Corte´s-Ortiz Museum of Zoology, Ecology and Evolutionary Biology, cruz, Tabasco, Oaxaca and Chiapas. As a result, this sub- University of Michigan, 1109 Geddes Ave., Ann Arbor, MI species is currently listed as critically endangered by the 48109, USA IUCN (Cuaro´n et al. 2008). Nevertheless, despite the fact that genetic management can be critical to the conservation L. A. Knapp Department of Anthropology, University of Utah, 270 S. 1400 of threatened taxa (McNeely et al. 1990), there have been E., Salt Lake City, UT 84112, USA no studies of genetic diversity in A. p. mexicana. 123 156 Primates (2014) 55:155–160

Recent assessments of the distribution of Mexican pri- visually checked for accuracy using MEGA 5 (Tamura mates have highlighted the importance of the Selva Zoque et al. 2011). In all cases, sequences were confirmed by (Zoque Forest), which lies at the border of the states of obtaining more than one copy from more than one extract Oaxaca, Chiapas, Tabasco and Veracruz, as the key and through the use of a combination of the forward and remaining forest refuge for A. p. mexicana (Oropeza-Her- reverse PCR primers and two inside sequencing primers: nańdez and Rendoń-Hernańdez 2012). At over (1) How-S6 (Ascunce et al. 2003) and (2) How-S7PrIME, 1,000,000 ha, the Selva Zoque is the largest remaining tract which we adapted from the How-S7 primer reported in of tropical rainforest in Mexico and is one of the most Ascunce et al. (2003) to be more specific to A. palliata important areas for biodiversity in Mesoamerica. A large samples as follows: 50-GGCGCTATCACCCTCTTAA-30. population of howler monkeys is assumed to remain in this We deposited all unique sequences in GenBank (accession region (Oropeza-Hernańdez and Rendoń-Hernańdez 2012). numbers KF725086–KF725092). However, to date there have been no surveys of this pop- We chose to amplify a relatively large sequence from ulation. Given the potential importance of this region for the faecal sample in order to minimise the probability of the conservation of A. p. mexicana, there is an urgent need encountering nuclear mitochondrial insertions (numts). As for demographic and genetic surveys. These data may be we detected a single PCR amplicon of the expected size in key to the future management and conservation of the all individuals, identified no extremely variant sequences subspecies. Here, we report the results of the first popula- and successfully aligned all sequences with a whole tion genetic survey of A. p. mexicana using faecal samples mitochondrial genome from Alouatta caraya (Finstermeier collected throughout the northern part of the Selva Zoque. et al. 2013), we consider that it is unlikely that our results include numts. We estimated genetic diversity of the mtDNA control Methods region sequences by calculating the number of haplotypes

In 2009 and 2010, we collected faecal samples from 45 wild howler monkeys, in 15 different sampling locations, in the northern part of the Selva Zoque, in the state of Veracruz (Fig. 1). Fresh faecal samples were collected from observable individuals immediately after defecation, making repeat sampling from the same individual extremely unlikely. Samples were collected in 15-ml universal tubes with 10 ml of 99 % ethanol and stored at room temperature. Samples were later transported to the Primate Immunogenetics and Molecular Ecology Laboratory at the University of Cambridge for processing and analysis. We successfully extracted DNA using the QIAamp DNA Stool Mini Kit (Qiagen, Germany) and sequenced the entire mitochondrial control region (1,100 bp) for all individuals. Polymerase chain reaction (PCR) ampliﬁcation was carried out using the universal mammalian control region primers H00651 and L15926 (Kocher 1989). The total PCR reaction volume was 25 ll, consisting of 8.0 ll DNA template, 2.5 ll PCR buffer (59, Bioline), 1.0 llof

MgCl2 (50 mM, Bioline), 2.0 ll of dNTPs (2.5 mM, Bio- line), 1.0 ll BSA (1 mg/ml), 0.4 ll of each primer (25 lM), 0.2 ll Taq polymerase (Bioline) and 10 ll ultrapure H2O. PCR conditions consisted of one denatur- ation step at 94 °C for 5 min, followed by 40 cycles of 94 °C for 60 s, 53 °C for 60 s, and 72 °C for 90 s. The reaction was then held at 72 °C for 10 min, before being stored at 4 °C until required for further analysis. Bidirectional sequences were generated from each PCR product from a commercial sequencing service (Macrogen Fig. 1 Map showing the positions of 15 sampling locations in the Inc., Korea) and aligned using the Clustal W function and northern part of the Selva Zoque, in Veracruz State, Mexico 123 Primates (2014) 55:155–160 157

(n), haplotype diversity (h), and nucleotide diversity (p)in Results Arlequin 3.5 (Excoffier and Lischer 2010) and compared these values to previously published data for other New We identified 7 different mtDNA control region haplotypes World primate species. To evaluate the extent of popula- with just 7 variable sites in the 45 samples collected for this tion subdivision we performed an analysis of molecular study (Table 1). Of these, 32 individuals (71.1 %) shared variance (AMOVA) and computed a general FST value in the same haplotype, 11 (24.4 %) individuals possessed one Arlequin 3.5 (Excoffier and Lischer 2010), using the 11 of 5 haplotypes that differed from the most common hap- sampling locations with two or more individuals as the lotype by 1 nucleotide substitution, and 2 (4.4 %) indi- units of our analysis. viduals possessed a single haplotype that differed from the We then used the control region sequences to infer most common haplotype by 3 nucleotide substitutions patterns of demographic history. Firstly, we applied (Table 1). The geographic distribution of the haplotypes is tests based on the distribution of segregating sites shown in Table 1. Results from the AMOVA suggest sig- (Tajima’s D; Tajima 1989) and haplotype distribution nificant population structuring in the samples collected:

(Fu’s Fs;Fu1997). Negative results of these tests are 87.01 % of the total molecular variance was found among indicative of a population that has undergone a recent sampling locations, whereas 12.99 % was found within expansion, as rare alleles are more common than sampling locations. Accordingly, the ﬁxation index was expected. Positive values occur when rare alleles are high and signiﬁcant (FST = 0.87, p \ 0.001). eliminated from a population following a bottleneck. Compared to previous studies of howler monkeys and

We tested for the signiﬁcance of D and Fs by gener- other New World primates, genetic diversity was extremely ating 10,000 coalescent simulations in Arlequin 3.5 low: h ± SD = 0.486 ± 0.088; p ± SD = 0.0007 ±

(Excofﬁer and Lischer 2010). As Fs is thought to be a 0.0002 (Table 2). Both standard tests of neutrality were more sensitive indicator of population expansion than negative and signiﬁcant: Tajima’s D =-1.48, p = 0.05;

D (Fu 1997), we only regarded Fs as significant if Fu’s Fs =-3.33, p = 0.02. The mismatch distribution p B 0.02. was unimodal and non-significant (SSD = 0.006, p = To further investigate demographic history, we analysed 0.38; r = 0.12, p = 0.33) (Fig. 2). the distribution of pairwise differences (mismatch distribution) for all sequences using Arlequin 3.5 (Excoffier and Lischer 2010). We used two statistics to test for demo- Discussion graphic expansion: the sum of squares deviation (SSD) and the raggedness index (r). The SSD tests the validity of a Compared to other howler monkey species, and other sudden expansion model based on the sum of squares Neotropical primates, we found extremely low genetic deviations between the observed and expected mismatch. diversity in the howler monkey population studied Non-significant SSD results indicate that the data do not (Table 2). These results may reflect a general trend in the deviate from that expected under expansion. A non-sig- Selva Zoque region as: (1) the habitat is largely continuous nificant raggedness index also indicates population in the region and the fragmentation history is relatively expansion. recent and (2) the distance between some of our samples

Table 1 Nucleotide sequence differences and geographic distribution of Mexican howler monkeys’ control region haplotypes in the Selva Zoque, Mexico

Haplotype Nucleotide position of variable sites Sampling locationa N

180 221 311 331 364 534 763 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 A AAAGGG3 11213827 2 232 2 G ...A.A 22 3 . ....A. 4 4 4 . .G.... 4 4 5 . G..... 1 1 6 . ..G... 1 1 7 ...... A 11 Total 3 4 1 1 2 5 3 8 2 71122345 a Sampling locations are shown in Fig. 1

123 158 Primates (2014) 55:155–160

Table 2 Mitochondrial DNA diversity in the studied population of Mexican howler monkeys and a range of New World primate species Species/subspecies N bp Regiona nh p References IUCN category

Alouatta caraya 73 &800 CR 34 0.946 0.0088 Ascunce et al. (2007) Least concern Alouatta caraya 44 1,140 cyt b 17 0.877 0.0023 Nascimento et al. (2007) Least concern Alouatta belzebul 66 1,140 cyt b 32 0.950 0.0100 Nascimento et al. (2008) Vulnerable Alouatta guariba clamitans 19 1,200 cyt b 8 – 0.0078 Harris et al. (2005) Least concern Brachyteles hypoxanthus 152 366 CR 23 0.905 0.0135 Chaves et al. (2011) Critically endangered Cebus albifrons 118 696 COII 55 0.944 0.0319 Ruiz-Garcı´a et al. (2010) Least concern Aotus azarai azarai 118 1,099 CR 30 0.830 0.0050 Babb et al. (2011) Least concern Alouatta palliata mexicana 45 1,100 CR 7 0.486 0.0007 This study Critically endangered N sample size, bp base pairs, n number of haplotypes, h haplotype diversity, p nucleotide diversity a The mtDNA control region (CR) evolves more rapidly than the cytochrome b (cyt b) and COII genes and, thus, would be expected to reveal higher population level diversity

arguing a final colonization as recent as the last glacial period (Corte´s-Ortiz et al. 2003). Reductions in population size due to bottlenecks during the expansion through Central America may have resulted in lower genetic diversity. Our results support this hypothesis, as we found significant deviations from constant population models of evolution and a unimodal mismatch distribution, both of which suggest the population has undergone a recent expansion (Fu 1997; Harpending 1994; Tajima 1989). However, these results should be interpreted with caution given: (a) the limited sample size and geographic distribution of the samples; (b) the borderline p values obtained in the statistical tests; and (c) the limited reliability of these types of tests (e.g., Yang et al. 2012). Examples from some mammals have shown that his- Fig. 2 The distribution of pairwise differences (mismatch distribution) of Alouatta palliata mexicana mitochondrial control region torically low genetic diversity is not necessarily a long- sequences term threat to the viability of species (Rodrı´guez et al. 2011; Miller et al. 2012; Tsangaras et al. 2012). However, was considerable (maximum distance = 96 km). We also loss of genetic diversity can reduce the ability of popula- found evidence of genetic structure in the population, tions to adapt to environmental change, as well as reducing which is likely a reflection of the unique haplotypes found reproductive fitness and disease resistance (Reed and in some sampling areas. However, given the small number Frankham 2003). Therefore, the maintenance of genetic of samples collected at each sampling location (sometimes diversity is one of the primary objectives in the manage- represented by just two individuals), these results should be ment of threatened species (McNeely et al. 1990). Given interpreted with caution. Nonetheless, the small sample that the Selva Zoque is by far the largest remaining forested size would be more likely to lead to the failure to detect area in the range of the subspecies, and that this subspecies population structure (Type 1 error) than to falsely indicate is currently listed as critically endangered by the IUCN population structure. (Cuaroń et al. 2008), this may present a worrying scenario Low genetic diversity may not be indicative of recent for the future conservation of A. p. mexicana. inbreeding or founder effects, but may reflect the biogeo- The mitochondrial control region is non-coding and is graphical history of the subspecies. Howler monkeys are the most polymorphic region of the mitochondrial genome. hypothesised to have a recent history in Mesoamerica, first Therefore, it is commonly used for intraspecific population expanding from south to north after the completion of the level studies (Frankham et al. 2010). Although studies Panama land bridge around 3 million years ago (Corte´s- focusing on single locus genetic markers may be limited in Ortiz et al. 2003; Ford 2006). Mantled howler monkeys scope, the mitochondrial control region has been the focus (A. palliata) are hypothesised to have colonised Mexico of many high-profile studies in wild primates. While many within the last million years (Ford 2006), with some of these studies have focused on just one of the two short

123 Primates (2014) 55:155–160 159 hyper-variable segments (HVI and HVII) of the control Argentinean Chaco. Am J Phys Anthropol 146:209–224. doi:10. region (e.g., Jensen-Seaman and Kidd 2001; Ascunce et al. 1002/ajpa.21567 Chaves PB, Alvarenga CS, Possamai CDB et al (2011) Genetic 2003; Liu et al. 2007; Chaves et al. 2011), the present study diversity and population history of a critically endangered analysed full-length control region sequences and still primate, the northern muriqui (Brachyteles hypoxanthus). PLoS found low diversity. An analysis of HVI would have ONE 6:e20722. doi:10.1371/journal.pone.0020722 yielded 6 haplotypes for this study, but if we examined Corte´s-Ortiz L, Rodrı´guez-Luna E, Sampaio I, Ruiz-Garcıá M (2003) Molecular systematics and biogeography of the Neotropical HVII alone we would have only encountered 2 haplotypes. monkey genus, Alouatta. Mol Phylogenet Evol 26:64–81 This highlights the value of using full-length control region Cuaroń A, Shedden A, Rodrı´guez-Luna E et al (2008) IUCN Red List sequences for population genetic analyses. of Threatened Species. Version 2012.2. http://www.iucnredlist. Future studies should use a range of mitochondrial and org (downloaded 5 April 2013) Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series nuclear markers to fully evaluate genetic diversity in A. p. of programs to perform population genetics analyses under mexicana. Such studies should be undertaken throughout Linux and Windows. Mol Ecol Resour 10:564–567. doi:10.1111/ its geographic range in order to evaluate population j.1755-0998.2010.02847.x structure and identify management units for conservation. Finstermeier K, Zinner D, Brameier M et al (2013) A mitogenomic phylogeny of living primates. PLoS ONE 8:e69504. doi:10.1371/ Due to the limited distribution and population size of A. p. journal.pone.0069504 mexicana, future conservation strategies may need to Ford SM (2006) The biogeographic history of Mesoamerican consider genetic management. Connecting currently exist- primates. In: Estrada A, Garber PA, Pavelka MSM, Luecke L ing forest fragments may be of benefit, as howler monkeys (eds) New perspectives in the study of Mesoamerican primates: distribution, ecology, behavior, and conservation. Springer, New are known to make use of corridors and live fences. York, pp 81–114 Translocation of some individuals or groups might also Frankham R, Ballou J, Briscoe D (2010) Introduction to conservation increase genetic diversity and minimise the risk of sto- genetics, 2nd edn. Cambridge University Press, Cambridge, chastic events, such as forest fires or disease, which could p 644 Fu Y-X (1997) Statistical tests of neutrality of mutations against lead to the local extinction of populations. This type of population growth, hitchhiking and background selection. strategy has been successfully implemented for A. p. Genetics 147:915–925 mexicana (e.g., Aguilar-Cucurachi et al. 2010). However, a Harpending H (1994) Signature of ancient population growth in a more detailed knowledge of the population genetics of the low-resolution mitochondrial DNA mismatch distribution. Hum Biol 66:591–600 subspecies is urgently recommended to maximise the Harris EE, Gifalli-iughetti C, Braga ZH, Koiffmann CP (2005) conservation impact of these strategies. Cytochrome b sequences show subdivision between populations of the brown howler monkey (Alouatta guariba) from Rio de Acknowledgments We gratefully acknowledge the National Janeiro and Santa Catarina, Brazil. Neotrop Primates 13:17–21. Council of Science and Technology (CONACYT) in Mexico and the doi:10.1896/1413-4705.13.2.16 Veracruz State Government for providing financial support to the Jensen-Seaman MI, Kidd KK (2001) Mitochondrial DNA variation project (Grant Number 108990). JD was supported by grants from the and biogeography of eastern gorillas. Mol Ecol 10:2241–2247 Isaac Newton Trust and the Cambridge Humanities Research Grant Kocher TD (1989) Dynamics of mitochondrial DNA evolution in Scheme. LCO is supported by an NSF grant (# BCS-0962807). We animals: amplification and sequencing with conserved primers. thank the Secretariat of Environment and Natural Resources in Proc Natl Acad Sci 86:6196–6200. doi:10.1073/pnas.86.16. Mexico and the Department for Food and Rural Affairs in the UK for 6196 providing transport permits for the faecal samples. Liu Z, Ren B, Wei F et al (2007) Phylogeography and population structure of the Yunnan snub-nosed monkey (Rhinopithecus bieti) inferred from mitochondrial control region DNA sequence analysis. Mol Ecol 16:3334–3349. doi:10.1111/j.1365-294X. 2007.03383.x References McNeely JA, Miller KR, Reid WV, Mittermeier, Russell A, Werner TB (1990) Conserving the world’s biological diversity. IUCN, Aguilar-Cucurachi MAS, Dias PAD, Rangel-Negrıń A et al (2010) Gland, Switzerland; WRI, Washington, DC; Conservation Preliminary evidence of accumulation of stress during translo- International; WWF-US; World Bank, pp 193. ISBN cation in mantled howlers. Am J Primatol 72:805–810. doi:10. 0-915825-42-2 1002/ajp.20841 Miller W, Hayes VM, Ratan A et al (2012) Genetic diversity and Ascunce MS, Corte´s-Ortiz L, Mudry MD (2003) The mitochondrial population structure of the endangered marsupial Sarcophilus control region of the black howler monkey, Alouatta caraya harrisii (Tasmanian devil). Proc Natl Acad Sci 109:18625. (Primates, Platyrrhini), and the development of new primers. doi:10.1073/pnas.1217345109 Mol Ecol Notes 15926:372–375. doi:10.1046/j.1471-8286.2003. Nascimento FF, Bonvicino C, Seuańez HN (2007) Population genetic 00454.x studies of Alouatta caraya (Alouattinae, Primates): inferences on Ascunce MS, Hasson E, Mulligan CJ, Mudry MD (2007) Mitochon- geographic distribution and ecology. Am J Primatol drial sequence diversity of the southernmost extant New World 1104:1093–1104. doi:10.1002/ajp monkey, Alouatta caraya. Mol Phylogenet Evol 43:202–215. Nascimento FF, Bonvicino CR, Oliveira MMDE et al (2008) doi:10.1016/j.ympev.2006.10.004 Population genetic studies of Alouatta belzebul from the Babb PL, Fernandez-Duque E, Baiduc CA et al (2011) mtDNA Amazonian and Atlantic forests. Am J Primatol 431:423–431. diversity in Azara’s owl monkeys (Aotus azarai azarai) of the doi:10.1002/ajp.20507

123 160 Primates (2014) 55:155–160

Oropeza-Hernańdez P, Rendoń-Hernańdez E (2012) Programa de MSM, Luecke L (eds) New perspectives in the study of accioń para la conservacioń de las especies: primates, mono Mesoamerican primates: distribution, ecology, behavior, and aranã (Ateles geoffroyi) y monos aulladores (Alouatta palliata, conservation. Springer, New York, pp 29–79 Alouatta pigra). SEMARNAT/CONANP, Mexico, pp 53. ISBN Tajima F (1989) The effect of change in population size on DNA 978-607-8246-10-6 polymorphism. Genetics 123:597–601 Reed DH, Frankham R (2003) Correlation between fitness and genetic Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular diversity. Conserv Biol 17:230–237. doi:10.1046/j.1523-1739. evolutionary genetics analysis using maximum likelihood, 2003.01236.x evolutionary distance, and maximum parsimony methods. Mol Rodrı´guez R, Ramı´rez O, Valdiosera CE et al (2011) 50,000 years of Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121 genetic uniformity in the critically endangered Iberian lynx. Mol Tsangaras K, A´ vila-Arcos MC, Ishida Y et al (2012) Historically low Ecol 20:3785–3795. doi:10.1111/j.1365-294X.2011.05231.x mitochondrial DNA diversity in koalas (Phascolarctos cinereus). Ruiz-Garcıá M, Castillo MI, Va´squez C et al (2010) Molecular BMC Genet 13:92. doi:10.1186/1471-2156-13-92 phylogenetics and phylogeography of the white-fronted capuchin Yang M, Yang Y, Cui D et al (2012) Population genetic structure of (Cebus albifrons; Cebidae, Primates) by means of mtCOII gene Guizhou snub-nosed monkeys (Rhinopithecus brelichi)as sequences. Mol Phylogenet Evol 57:1049–1061. doi:10.1016/j. inferred from mitochondrial control region sequences, and ympev.2010.09.002 comparison with R. roxellana and R. bieti. Am J Phys Anthropol Rylands AB, Groves CP, Mittermeier RA et al (2006) Taxonomy and 147:1–10. doi:10.1002/ajpa.21618 distribution of Mesoamerican primates. In: Garber PA, Pavelka

123