The Y-chromosome landscape of the Philippines: extensive heterogeneity and varying genetic affinities of Negrito and non-Negrito groups
Frederick Delfin1,2, Jazelyn M. Salvador1, Gayvelline C. Calacal1, Henry B. Perdigon1, Kristina A. Tabbada1, Lilian P. Villamor1, Saturnina C. Halos1, Ellen Gunnarsdóttir2, Sean Myles1,6, David A. Hughes2, Shuhua Xu3, Li Jin3, Oscar Lao4, Manfred Kayser4, Matthew E. Hurles5, Mark Stoneking2 and Maria Corazon A. De Ungria1*
1DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, 1101, Quezon City, Philippines;
2Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz, D04103, Leipzig, Germany;
3Chinese Academy of Sciences and Max Planck Society Partner Institute for Computational Biology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031 China;
4Department of Forensic Molecular Biology, Erasmus University Medical Center Rotterdam, Dr. Molewaterplein 50, 3000 CA Rotterdam, The Netherlands;
5The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom;
6Current affiliation: Institute for Genomic Diversity, Cornell University, Ithaca, NY 14853-2703 USA.
*Corresponding author: Maria Corazon A. De Ungria, PhD
DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, 1101, Quezon City, Philippines. Telefax: (63-2) 925-2965, E-mail:
SUPPLEMENTARY TEXT: MATERIALS AND METHODS
POPULATION SAMPLE HISTORY
The 390 samples included in this study comprise two sets of population sample collections. One set, initially composed of 1032 samples (607 males, 425 females) representing 15 Filipino language groups (excluding Surigaonon) (Figure 1) was the result of population sampling efforts by the University of the Philippines, Natural Sciences Research Institute DNA Analysis Laboratory (UP-NSRI-DAL) from 1997–2004. Biological samples in this collection consisted of whole blood blotted on FTA? cards (FTA? Gene Guard system, Whatman Inc., Springfield Mill, Maidstone, Kent, UK), buccal cells collected by swab and transferred on FTA? cards and plain buccal swabs. The FTA? Gene Guard system (Whatman Inc., Springfield Mill, Maidstone, Kent, UK) was used to extract DNA from whole blood blotted on FTA? paper and from saliva-buccal cells swabbed on FTA? paper.1 A phenol-chloroform method2 and/or the QIAamp? DNA Blood Mini kit (QIAGEN Inc., Valencia, CA, USA) were used to extract DNA from buccal swab samples. DNA samples were stored in the UP-NSRI-DAL sample repository. Given the length of time in storage, DNA samples from some groups (Ivatan, Bugkalot, Kalangoya, CAR and Maranao) required enrichment and were therefore subjected to whole genome amplification (WGA) using the GenomiPhi? DNA amplification kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) kit following manufacturer’s instructions. Male DNA samples in this collection were typed for Y-chromosome binary markers (Y-SNPs) at the Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K. following a protocol adopted from the work of Paracchini, Hurles and colleagues3,4 with modifications (Supplementary information: Figure 1, Table 1, Text – DNA typing) using the ABI Prism? SNaPshot? multiplex kit (Applied Biosystems, Foster, CA, USA) with amplicons detected using capillary electrophoresis (CE) on an ABI Prism? 3100 Genetic Analyzer (Applied Biosystems, Foster, CA, USA) following manufacturer's instructions.
Another set of 142 samples (86 males, 56 females) consisted of saliva samples from the Mamanwa, Manobo and Surigaonon groups (Figure 1) collected by the Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany in 2005. Saliva samples were processed at MPI-EVA, using a high-salt, DNA extraction method.5 Male DNA samples in this collection were typed for Y-SNPs at the MPI-EVA, using an in-house validated, single-base extension (SBE) assay (Supplementary information: Figure 1, Table 1, Text – DNA typing) with amplicons detected using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) as described elsewhere.6
Male DNA samples of both the UP-NSRI-DAL and the MPI-EVA were typed for Y-chromosome microsatellite (Y-STR) markers at the UP-NSRI-DAL, Philippines using the PowerPlex? Y system (Promega Corporation, Madison, WI, USA) with amplicons detected on an ABI Prism? 310 Genetic Analyzer (Applied Biosystems, Foster, CA, USA), all following the manufacturers’ instructions.
Given that the Mamanwa and Manobo groups were independently sampled by the UP-NSRI-DAL and the MPI-EVA from the same Philippine region (Figure 1), sample overlap and/or relatedness were checked using genealogical information. All samples were also typed for autosomal microsatellite (A-STR) markers using the PowerPlex? 16 system (Promega Corporation, Madison, WI, USA) with amplicons detected on an ABI Prism? 310 Genetic Analyzer (Applied Biosystems, Foster, CA, USA), all following manufacturers’ instructions; and as such, Mamanwa and Manobo A-STR genotypes were also used to check for sample overlap and/or relatedness.
After excluding related and/or overlapping Mamanwa and Manobo samples; as well as DNA samples that did not produce reliable results after Y-SNP and Y-STR typing, the UP-NSRI-DAL male sample set (n=607) and the MPI-EVA male sample set (n=86) were reduced to 317 and 73, respectively; resulting in 390 samples that were included in this study (Figure 1).
POPULATION SAMPLING AND SAMPLE DETAILS
Population field sampling was conducted within enclaves (sitios) and/or districts (barangays) found within municipalities or cities across the Philippines. Before the collection of samples, interviews were conducted to compile the family history of each potential participant. Samples were generally collected from unrelated volunteers. However in some populations with small population sizes, most individuals were related; hence volunteers not related within the second degree of consanguinity were considered. A total of 1174 (1032: UP-NSRI-DAL; 142: MPI-EVA) population samples were collected. The following details of population sample collection enumerate the number of collected samples, sample type, collection sites and various personalities, institutions and organizations who assisted in sample collection from 1997 to 2005.
UP-NSRI-DAL collection
The CAR (Cordillera Administrative Region) group in this study is composed of three language groups namely: the Ibaloi (buccal swabs from 19 males and 32 females) from Benguet and Nueva Vizcaya provinces; the Ifugao (buccal swabs from 28 males and 26 females) from Banaue municipality in Ifugao province, Nagtipunan municipality in Quirino province and Kayapa municipality of the Nueva Vizcaya province; and Kankanaey (buccal swabs from 13 males and 50 females) from Mankayan and La Trinidad, Benguet province. Samples were collected during several trips to the CAR region in 1997 and 2002. After DNA typing, only two Ibaloi, one Ifugao and six Kankanaey samples gave reliable results. Due to close geographical proximity and cultural relatedness of the Ibaloi, Ifugao and Kankanaey language groups, nine samples were pooled as one sample set and labeled as the CAR group. Dr. Michael Paul Tan and Maricar Posa were involved in the collection of samples in 1997. Armando Bogite and members of the New Tribes Mission facilitated the collection of samples in 2002.
Aeta of Zambales samples (19 male and 19 female whole blood samples) were collected in January 1998 from volunteers who reside in the municipality of Castillejos, located north of the Subic Bay Metropolitan Authority (SBMA), in the province of Zambales. The collection was facilitated by Dr. Edith Tria of the Department of Health and Dr. Leo Uy.
Bugkalot samples (31 male and 21 female saliva samples) were collected from volunteers residing in different municipalities of Nueva Vizcaya and Quirino provinces in two separate trips in January and February, 2002, respectively. The collection of samples was facilitated by Rey and Lea de la Rosa, Pastor Angel de la Cruz and Jessie Magallanes, all from the New Tribes Mission and Armando Argayosa. Ramon Pagsiguian accompanied the sampling team to the community while Liza Faustino and Laarni Carpina assisted in the collection of samples.
Ivatan samples (162 male and 104 female buccal swabs) were collected from volunteers from the main islands of Batanes province namely Batan, Itbayat and Sabtang in March 2002 through the assistance of Leticia Bayaras (Provincial Health Office), Hilda Dacles (Municipal Health Office) and Patricia Gallo of the National Commission on Indigenous Peoples (NCIP) with endorsement from Governor Vicente Gato and facilitated by Dr. Victor Paz of the UP Archaeological Studies Program. Laarni Carpina assisted in the collection and handling of samples.
Kalangoya samples (45 male and 15 female buccal swabs) were collected from volunteers from Bambang and Kayapa municipalities of Nueva Vizcaya province in March 2002 with the assistance of Satur Banih and with support from Mayor Tony Dupiano, Jimmy Tindaan and Steve Baccar.
Maranao samples (28 male and 21 female buccal swabs) were collected in April 2002 from volunteers in two campuses of Mindanao State University (MSU), namely MSU-Iligan Institute of Technology (MSU-IIT) in Lanao del Norte province and MSU-Marawi, Marawi City, Lanao del Sur province. Permission to collect on campus was obtained from Dr. Olga Nuneza of MSU-IIT. Irene Estrada Macarambon- Benito of MSU-IIT facilitated volunteer participation in the study.
Aeta of Bataan samples (27 male and 14 female samples of saliva blotted on FTA? paper) were collected in April 2003 from volunteers residing in the municipalities of Morong, Hermosa and Orion in the province of Bataan and in the area governed by the SBMA in the Zambales province through the assistance of Edmond de Jesus and with the endorsement of Amethya Concepcion of the SBMA, Dr. Roberto Pagulayan of the UP Institute of Biology, Tribal Chief Bonifacio Florentino and Tribal Chief Josefina Alejo. Joseph Salonga accompanied the sampling team to the communities. The Aeta of Bataan is differentiated from the Aeta of Zambales because these groups use different languages.7
Filipino groups residing on the island of Mindoro are collectively called Mangyan. Samples of saliva blotted on FTA? paper were collected from four Mangyan groups namely the Hanunuo (15 male and 3 female samples), Iraya (16 male and 11 female samples), Tadyawan (14 male and 1 female samples) and Tawbuid (14 male and 1 female samples) in April 2003 during a gathering organized by the Mangyan Church Tribal Association (MCTA) in Baco municipality and Calapan city of Oriental Mindoro province. Pastor Andino Layda, Efren Aceveda, Peter Mayot and Pastor Diokno Onday of the MCTA and Tribal leader Fundador Fuentes assisted in obtaining the free and prior informed consent (FPIC) from volunteers. John Richards, Lore Jean de Guito and Dothy Smith of the Overseas Mission Fellowship assisted in introducing the sampling team to the community.
Ati samples (36 male and 26 female samples of saliva blotted on FTA? paper) were collected from volunteers from different districts of the provinces of Antique, Aklan, Capiz and Iloilo, all on the island of Panay as well as from the adjacent islands of Boracay and Guimaras in January 2004. Sampling was coordinated with Pastor Emeterio Allianza, Pastor Jessie Elosendo, Pastor Enoc Valencia, Chief Salvador Escu?a, Chief Gregorio Elosendo, Chief Elias Valencia and Chief Enrique Martinez. Alejandro Condez accompanied the sampling team around Panay and Guimaras islands whereas Joana Guarin of the Aklan State University helped in the collection of samples from residents on the island of Boracay.
Agta samples (44 male and 23 female samples of saliva blotted on FTA? paper) were collected from volunteers residing in Iriga City and Buhi municipality, both within the province of Camarines Sur in March 2004. Contact with the communities was made with the assistance of Julio Versoza of the Mt Isarog Protected Area Office, Dennis Barroga and Belen Jacob of the National Commission on Indigenous People (NCIP). Our request to go to the communities was approved by Director Lee Arroyo and Atty. Corazon Crescini of the NCIP.
Mamanwa samples (26 male and 21 female samples of saliva blotted on FTA? paper) were collected from volunteers from different municipalities of the province of Surigao del Norte in April 2004, through the assistance of Leonita Gorgolon, Audie Reliquette and Angelita Bullo, from the Provincial Health Office and Pastor Bernard Yap of the Christ Faith Fellowship Church.
Manobo samples (70 male and 37 female samples of saliva blotted on FTA? paper) were collected from volunteers from different municipalities of the province of Agusan del Sur with the assistance of Mrs. Lily Labadan and Maria Marley Daday in April 2004.
MPI-EVA collection
In August 2005, samples of saliva in lysis buffer were collected from Mamanwa (38 male and 20 female samples); Manobo (11 male and 36 female samples) and Surigaonon (37 male samples) groups. Mamanwa and Surigaonon volunteers were from different municipalities of Surigao del Norte province and Manobo volunteers were from different municipalities of Agusan del Norte province. Population sampling was facilitated by Mr. Fernando A. Almeda Junior, Dr. Irinetta C. Montinola, Dr. Wilfredo Sinco (all from the Surigaonon Heritage Center); Ms. Girlie Patagan (NCIP); Mrs. Elizabeth S. Larase and Ms.Juliet P. Erazo (Office of Non-Formal Education) and the Rotary Club of Surigao.
DNA TYPING
Multiplexes I.1 to III.19 (Supplementary Information: Figure 1 and Table 1)
Specific-PCR Multiplex reactions were performed in 10 microliter (μl) volumes each containing 1X AmpliTaq Gold Buffer II (Applied Biosystems, Foster, CA, USA), 4 millimolar (mM) Magnesium Chloride (MgCl2), 0.4 mM deoxyribonucleotide triphosphate mix (dNTP mix), 0.08–0.24 μM primer mix (Supplementary Table 1), 0.5 Unit AmpliTaq Gold? enzyme (Applied Biosystems, Foster, CA, USA) and 2 μl or 1 FTA? punch for DNA template; with the following thermocycling parameters: 94oC for 9 minutes (min); 15 cycles of 94oC for 30 seconds (sec), 59oC for 30 sec, 72oC for 60 sec and a final 72oC for 3 min.
With the incorporation of universal tags or “ZIP code” sequences (Supplementary Table 1)3 in the specific-PCR products, even concentration of specific-PCR products in the multiplex was obtained through a second amplification using high concentrations of “ZIP code” primers. These reactions were performed in 20 μl volumes each containing 1X AmpliTaq Gold Buffer II (Applied Biosystems, Foster, CA, USA), 4 mM MgCl2, 1 μM each of ZIP code primer A and primer B (Supplementary Table 1), 0.5 Unit AmpliTaq Gold? enzyme (Applied Biosystems, Foster, CA, USA) and 10 μl specific PCR product; with the following thermocycling parameters: 94oC for 9 min; 30–34 cycles of 94oC for 30 sec, 55oC for 30 sec, 72oC for 60 sec and a final 72oC for 3 min.
PCR products (specific PCR-ZIP reaction product) were cleaned in 12 μl-volume reactions each containing 2 Units of shrimp alkaline phosphatase (SAP) enzyme, 1.5 Units of Exonuclease I (Exo I) enzyme and 10 μl PCR product with the following incubation parameters: 37oC for 1 hour (hr) and 80oC for 20 min.
Single-base extension (SBE) reactions were performed in 5 μl volumes using the ABI Prism? SNaPshot? multiplex kit (Applied Biosystems, Foster, CA, USA) following manufacturer's instructions. Each SBE reaction contained 1X SNaPshot? reaction mix, 0.2–0.6 μM extension primer mix (Supplementary Table 1) and 0.5 μl of cleaned PCR product with the following thermocycling parameters: 25 cycles of 96oC for 10 sec, 50oC for 5 sec and 60oC for 30 sec. SBE products were detected by CE on an ABI Prism? 3100 Genetic Analyzer (Applied Biosystems, Foster, CA, USA) following manufacturer's instructions.