SYLVATROP Editorial Staff

Leuvina M. Tandug Editor-in-Chief Eliseo M. Baltazar Adreana Santos-Remo Managing Editor Jobelle Mae L. Zuraek Layout/Graphics Editor Liberty E. Asis Adreana Santos-Remo Eduardo M. Tolentino Editors Circulation Assistant

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January - December 2013 Vol. 23 Nos. 1&2

SYLVATROP, The Technical Journal of Philippine Ecosystems and Natural Resources is published by the Department of Environment and Natural Resources (DENR) through the Ecosystems Research and Development Bureau (ERDB), College, Laguna.

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Cover Photo: Portion of the Paraiso Reforestation Project in Barangay Tangaoan, Piddig, Ilocos Norte manifesting tree species diversity 80 years after its establishment in 1930. (Courtesy of For. Manolito U. Sy, ERDB)

Cover Layout: Jobelle Mae L. Zuraek Sylvatrop, The Technical Journal of Philippine Ecosystems and Natural Resources 22 (1 & 2): 1 - 20

Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites in Los Baños, Laguna and Quezon City, Philippines

Arcely C. Viernes Science Research Specialist II Research and Development Division, Environmental Management Bureau DENR Compound, Visayas Avenue, Diliman, Quezon City Tel. No.: 632-426-4332 Email address: [email protected]

Maricris T. Laciste Science Research Specialist II

This study aimed to establish trends of levels of acidity (pH), conductivity (EC), and 2- - - + + + 2+ 2+ concentrations of anions (SO4 , NO3 , Cl ) and cations (NH4 , Na , K , Ca , Mg ) in rainwater on urban and rural acid deposition monitoring sites in the Philippines. The results of the study will be significant in establishing local acid deposition baseline data to prove or disprove acid deposition occurrence in the Philippines and to provide useful inputs for decision-making aimed at preventing or reducing adverse impacts of acid deposition. The investigation was conducted in Los Baños, Laguna (Los Baños site) and Quezon City (Metro Manila site), categorized as rural and urban sites, respectively, under the Acid Deposition Monitoring Network in East Asia (EANET) protocol. Weekly monitoring, collection, and laboratory analysis of samples were conducted using standard analytical methods and EANET quality assurance and quality control (QA/QC) procedures.Time series analysis of data from 2001 to 2009 for both sites showed a general decreasing annual trend for all parameters. Seasonal trends were observed to be consistent with annual trends. Correlations between the parameters analyzed are outside the scope of this study and should be the focus of further assessment of the state of acid deposition in the Philippines.

Keywords: pH, EC, anions, cations, acid deposition, rainwater, EANET 2 A. Viernes and M. Laciste

Measurements of precipitation chemistry have become increasingly important during the last four decades. These measurements provide information on the exchange of trace materials between the atmosphere and the earth's surface (EANET 2000). At an ambient carbon dioxide mixing ratio of 350 ppm and temperature of 298ºK, a raindrop in a pollutant-free atmosphere has a pH of 5.6 as a result of pure water in equilibrium with the atmospheric concentration of carbon dioxide (Seinfeld and Pandis 1998). However, anthropogenic activities resulting in emissions of acidic precursors in the atmosphere such as SO2 and NOx lower the pH of rainwater reaching the ground. On the other hand, atmospheric base cations and other crustal elements neutralize to a certain extent the atmospheric acidity contributed by anthropogenic emissions. The net acidity of the rainwater is the overall result of the acid and base contributions.

This article focuses on the generated data on wet deposition (rainwater) samples collected from the two EANET sites in the Philippines, namely the Metro Manila site in Quezon City and the Los Baños site in Laguna, representing urban and rural sites, respectively. Rainwater data from 2001 to 2009 were utilized to present the trends of the levels of acidity (pH), electric conductivity (EC), concentrations 2- - - + + of sulfate (SO4 ), nitrate (NO3 ), chloride (Cl ), ammonium (NH4 ), sodium (Na ), potassium (K+), calcium (Ca2+), and magnesium (Mg2+). The established trends are significant as baseline data to challenge acid deposition occurrence, both local and transboundary, and as useful inputs for decision-making and policy implementation aimed at preventing or reducing the adverse impacts of acid deposition on the environment and human health.

Review of literature

It was in the nineteenth century when people first observed signs of deterioration of forests in the vicinities of large industrial areas in England. Scientist Robert Angus Smith of England coined “acid rain” when he observed that acidic precipitation could damage plants and materials (US Environmental Protection Agency [USEPA] 2011). Acid rain, however, was only considered a serious environmental issue until the 1970s (EANET 2006).

Acid deposition, commonly called acid rain, is a result of the combustion of fossil fuels through industrial processes and transportation. The emissions of sulfur dioxide (SO2) and nitrous oxides (NOx) to the atmosphere undergo complex chemical reactions, and fall to the earth as wet deposition (rain, snow, fog, cloud) or dry deposition (dry particles, gas). Reactions of the two compounds with water, oxygen, carbon dioxide, and sunlight in the atmosphere result to sulfuric acid (H2SO4) and Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 3

nitric acid (HNO3), the primary sources of acid deposition. These acidic substances can travel thousands of kilometers from the original emission sources. Thus, acid deposition is not limited by national boundaries but has become a regional, as well as an international issue.

Acid deposition has adverse impacts on forest, soil, lakes, rivers, wildlife populations, historical structures, buildings, and human health. In Canada, the decline in fish numbers in the 1960s to 1970s was believed to be caused bythe increasing acidity of water. But scientists later discovered that the acidified lakes had high concentrations of toxic metals (aluminum, cadmium and mercury) that were leached from soils and rocks surrounding those lakes (Pidwirny & Jones 2009). Furthermore, acid deposition can leach several important plant nutrients, such as calcium, magnesium, and potassium, thereby weakening the plant structure.

Acid rain also accelerates the decay of structural materials and paints, including historical buildings, stone monuments, and sculptures. Also, acid rain adversely affects human health through the respiratory system. SO2 and NOx cause respiratory problems like asthma, dry coughs, and irritation of the throat and eyes.

The increasing threat of acid deposition due to increased emissions of air pollutants into the atmosphere has prompted the creation of the following regional networks of air pollution and acid deposition monitoring throughout the globe:

1. European Monitoring and Evaluation Programme (EMEP) started in 1977 (United Nations [UN] 2004), and in 1979, the Convention on Long-Range Transboundary Air Pollution (CLRTAP) was established with the objective “to limit and, as far as possible, to gradually reduce and prevent air pollution including long-ranged air pollution” (UN Economic Commission for Europe [UNECE] 2008); 2. National Acid Precipitation Assessment Program (NAPAP) was initiated by North America in 1980. As a consequence, the 1990 U.S. Clean Air Act included a special section on acid rain intended to reduce transboundary pollution to Canada (NAPAP 2005); 3. National Atmospheric Deposition Program (NADP) in North America and the Canadian Air and Precipitation Monitoring Network (CAPMoN) in Canada were created for acid deposition monitoring and research purposes; 4. International Global Atmospheric Chemistry Project - Deposition of Biogeochemically Important Trace Species (IGAC/DEBITS); 5. World Meteorological Organization Global Atmosphere Watch (WMO/ GAW) Program; and 6. Acid Deposition Monitoring Network in East Asia (EANET 2006). 4 A. Viernes and M. Laciste

EANET was established in 1998 with three objectives: (1) create a common understanding of the state of acid deposition problems in East Asia; (2) provide useful inputs for decision-makers at local, regional, and international levels, aimed at preventing or reducing adverse impacts on the environment caused by acid deposition; and (3) contribute to cooperative efforts among participating countries on issues related to acid deposition. EANET monitoring covers four environmental media - wet deposition, dry deposition, inland aquatic environment, and soil and vegetation. EANET published the first “Periodic Report on the State of Acid Deposition in East Asia” (PRSAD) in November 2006. The report presented the monitoring data generated on the four environmental media from 2001 to 2004.

- + A comparison of the relationship of the deposition of NO3 and NH4 among the monitoring networks showed that ammonium deposition was generally higher in the EANET region than in EMEP and NADP, but nitrates showed an opposite pattern (EANET 2006).

European precipitation and mean sulfate concentrations showed sharp declines since the mid-1980s (Seinfeld and Pandis 1998). According to Seinfeld and Pandis, “In both the western and eastern United States, the summer and winter pH spatial trends are distinctly different. In the East, summer pH values are lower (median in summer is 4.45 while in winter it is 4.64) based on 1987 data. In the East, summer sulfate rainwater concentration is nearly twice that in the winter deposition. In the Western United States, summer sulfate deposition is twice that of winter”.

Figure 1 shows the pH values of precipitation in China’s South East area. Compared to Figure 2, after around 12 years, the annual average pH values of precipitation in some areas in China have increased (became less acidic) but in other areas, the values have not changed.

During the last decade, Asia Pacific countries had the largest global coal consumption of which China had taken the bulk (Fig. 3). Likewise, this is true for energy consumption around the globe. Acidic emissions during 1970-2000 were of decreasing trends in Western and Eastern Europe, as well as in North America, but of increasing trend in East Asia, which is considered a fast growing region in the world as depicted in Figure 4.

The Philippines reduced sulfur content of industrial and automotive diesel fuel from 0.2 to 0.05 percent since January 2004 as part of the implementation of the Philippine Clean Air Act of 1999. Pure diesel (with 0.05% S) was made available by five new oil players since September 2003. The Biofuels Act of 2006 also requires that biofuels and their blends, either manufactured or imported, are sold conforming to the Philippine National Standards. Through public awareness and education on Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 5

pH>7.0 pH=4.5-5.0 pH=5.6-7.0 pH=4.0-4.5 pH=5.0-5.6 pH<4.0

Figure 1. pH values of rainwater in China in 1997 (Ohizumi 2006).

pH value range <4.5 4.5-5.0 5.0-5.6 >5.6 no data available

Isograms of annual average pH values of the precipitation in 2009 in China

Figure 2. Annual average pH values of rainwater in China in 2009 (Ministry of Environmental Protection The People’s Republic of China 2009). 6 A. Viernes and M. Laciste

3000

2500

2000

Total North America

1500 Total S. & Cent.America

Total Europe & Eurasia

Total Middle East

1000 Total Africa

Total Asia Pacific

500 Coal consumption, Million Tonnes Oil Equivalent Coal consumption, Million Tonnes 0

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

1800

Australia

1600 Bangladesh

China

1400 China Hongkong SAR India

1200 Indonesia Japan Malaysia 1000 New Zealand

Pakistan 800 Philippines

South Korea 600 Taiwan

Thailand 400 Vietnam

Other Asia Pacific 200 Coal consumption, Million Tonnes Oil Equivalent Coal consumption, Million Tonnes 0

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Figure 3. World total coal consumption in 2000-2010 (top chart) and Asia Pacific total coal consumption in 2000-2010 (bottom chart) (BP Statistical Review of World Energy 2011). Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 7 air quality management based on Republic Act No. 8749, otherwise known as the Philippine Clean Air Act of 1999, various activities were undertaken in partnership with different multi-stakeholders (e.g., academic institutions, business, local government units, national government agencies, non-government organizations, private sectors) (EMB 2009).

Philippines, as one of the first ten countries that participated in the establishment of the EANET, has been conducting monitoring of the levels of the abovementioned ten major parameters of rainwater (wet deposition). Monitoring data are evaluated locally and submitted annually to the Network Center, the Asia Center for Air Pollution Research in Niigata, Japan.

45,000

40,000

35,000 Western Europe

30,000 North America Eastern Europe

25,000 Asia

20,000 (unit 1,000) tons) 15,000

Source: "Global 10,000 sulfur emissions from 1850 to 2000." Stern, David Che- 5,000 mosphere 58 (2005) 163-175.

0 1970 1975 1980 1985 1990 1995 2000

Figure 4. Trend of SO2 emissions in Asia, Europe, and America (Ohizumi 2006). 8 A. Viernes and M. Laciste

Methodology

Description of study sites

The first two wet deposition monitoring sites of EANET in the Philippines are Metro Manila and Los Baños. The Metro Manila site is located at the Ateneo de Manila University in Quezon City, representing an urban site. Within 2-kilometer radius are the thoroughfares of Katipunan Avenue, Aurora Boulevard, and Marcos Highway with more than 5,000 vehicles per day. Around ten colleges and universities, commercial establishments, and some residential areas surround the site within a 10-kilometer radius. Within the 10-50 kilometer radius are two power plants, industrial zones, a number of subdivisions, the Laguna Lake, and Manila Bay.

The Los Baños site situated at the Philippine Atmospheric, Geophysical and Astronomical Services Administration (PAGASA) – Agrometeorological Station, University of the Philippines Los Baños, College, Laguna, represents a rural site. Within the 10-kilometer radius of the site are rice fields, residential areas, commercial establishments, national road, the Mt. Makiling Forest Reserve, and Laguna Lake. Within the 10-50 kilometer radius are five power plants, industrial zones, refinery and natural gas, the Manila Bay, and Batangas Bay (EANET 2006).

Materials and method

Since the preparatory phase of EANET, wet deposition monitoring has been conducted and can be found in the EANET 2000 Technical Documents for Wet Deposition Monitoring. Rainwater samples were collected weekly by wet-only samplers installed at the monitoring sites, and transported in ice-cold chests to the laboratory for analysis of the required parameters, namely pH, electric conductivity 2- - - (EC), anions [i.e., sulphate (SO4 ), nitrate (NO3 ) and chloride (Cl )], and cations [i.e., + + + 2+ ammonium (NH4 ), sodium (Na ), potassium (K ), calcium (Ca ), and magnesium (Mg2+)]. Samples were analyzed by the analysts at the Environmental Management Bureau Laboratory using EANET’s analytical methods (Table 1). QA/QC programs were carried out in all stages of the monitoring activities in the field and in the laboratory. Cations and anions balance, as well as the electric conductivity comparison between the calculated and measured conductivity of each sample, should fall within the set criteria for quality data. Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 9

Table 1. Analytical methods for wet deposition monitoring in the Philippines. Parameter Analytical method pH Glass Electrode EC Conductivity Cell 2- - - SO4 , NO3 , Cl Ion Chromatography + NH4 Spectrophotometry (Indophenol Blue) / Ion Chromatography Na+, K+, Ca2+, Mg2+ Atomic Absorption Spectrometry/ Ion Chromatography To achieve the objective of this study, the annual wet deposition monitoring data of the established Metro Manila and Los Baños monitoring sites from 2001– 2009 were consolidated. Annual means, as well as the seasonal means, of the levels of pH and EC and of the concentrations of ions in the samples were computed and compared with each site.

Results and discussion

During the 2001 to 2009 study period, a total of 278 samples were collected from Metro Manila (MM) and 309 from Los Baños (LB). The annual mean values of ionic concentrations and levels of electric conductivity and pH of the samples collected from Metro Manila and Los Baños are presented in Tables 2 and 3, respectively.

The succeeding figures show the trends of the major parameters analyzed within the study period. Using linear regression analysis, a best fit line with the corresponding linear equation was used to establish an approximate trend within the monitoring period.

Annual mean concentration trends of anions followed downward trends over time (Fig. 5). The anions of Los Baños site showed greater degrees of decreases than those of the Metro Manila site.

+ + + 2+ Likewise, ammonium (NH4 ), sodium (Na ), potassium (K ), calcium (Ca ), and magnesium (Mg2+) in both sites showed decreasing trends over time (Fig. 6). All cations, except for calcium in the urban site, exhibited greater regression slopes than those in the rural site. 10 A. Viernes and M. Laciste

Table 2. Annual mean concentrations of rainwater components in Metro Manila site.

2- - - + + + 2+ 2+ SO4 NO3 Cl NH4 Na K Ca Mg EC pH Year value µmol/L mS/m 2001 34.76 26.78 82.74 124.4 116.5 73.53 56.34 36.26 7.66 6.53 2002 40.63 21.77 24.69 145.8 20.68 32.89 20.48 7.32 4.02 6.43 2003 25.64 27.30 22.09 42.33 17.74 8.73 15.42 4.02 1.72 4.84 2004 23.71 17.27 26.85 45.19 19.64 4.46 12.62 5.52 1.75 5.61 2005 31.68 26.71 20.20 43.07 20.57 4.60 18.92 4.19 2.11 5.42 2006 27.30 18.65 30.84 45.94 31.04 19.98 24.30 7.42 2.44 5.84 2007 33.12 24.17 34.01 60.34 27.71 11.88 21.44 7.56 2.46 5.82 2008 34.73 27.41 34.04 86.51 31.70 30.33 26.81 7.43 4.77 6.26 2009 18.66 13.23 33.12 29.44 28.81 11.65 12.73 5.00 3.55 5.76

Table 3. Annual mean concentrations of rainwater components in Los Baños site.

2- - - + + + 2+ 2+ SO4 NO3 Cl NH4 Na K Ca Mg EC pH Year value µmol/L mS/m 2001 22.08 49.74 60.25 38.01 56.85 20.07 47.78 15.43 3.30 6.45 2002 28.96 9.88 72.27 79.98 38.28 14.74 11.24 7.72 2.18 6.40 2003 10.78 7.75 25.15 23.57 42.23 5.53 11.69 5.31 1.54 4.76 2004 10.28 7.46 22.54 19.91 18.07 3.02 6.87 4.46 1.06 5.51 2005 16.81 15.05 34.05 34.48 31.51 3.77 6.99 4.75 1.66 5.59 2006 12.42 9.45 32.37 18.59 33.60 2.93 7.13 4.19 1.38 5.82 2007 13.79 10.75 44.49 26.90 31.60 7.77 8.56 6.19 1.38 5.77 2008 15.29 9.65 29.85 16.17 26.20 2.38 5.15 5.02 1.66 5.38 2009 11.27 10.02 36.16 17.32 28.25 7.72 5.22 4.21 1.12 5.78 Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 11

Electric conductivity and pH values followed downward trends over time at both monitoring sites (Fig. 7). Higher levels of annual mean EC were observed at the urban site. The annual mean pH of precipitation at both sites in 2003 and at Los Baños in 2008 were <5.6, which were considered to be acidic. The rest of the annual mean pHs during the study period were not acidic.

The seasonal variations of the levels of anions, cations, and EC and pH measured at the urban and rural sites are presented in Figures 8-10, respectively.

In the wet season months of May to October, seasonal means of anions at both sites had little variations over time as shown by the slopes equations. 2- - Sulphate (SO4 ) and chloride (Cl ) at Metro Manila site showed downward slopes - but nitrate (NO3 ) showed an upward slope. At Los Baños site, all anions exhibited slightly upward trends.

+ + 2+ + The NH4 , K , and Mg at both sites, and Na at the rural site had little variations over time during wet season. As of the dry season, Ca2+ at both sites and Na+ at urban site were also of decreasing trends during the wet season. Generally, seasonal means were higher during the dry season than those during the wet season.

Electric conductivity means of both urban and rural sites had little variations as shown by the slopes (Fig. 10). Mean pH values of rainwater at Los Baños during wet season had little variations while those of Metro Manila exhibited an increasing trend.

During the dry season, from November to April, seasonal means of all the measured components at both sites exhibited negative slopes of decreasing trends over time.

The data from Metro Manila and Los Baños sites from 2001 to 2009 showed decreasing trends of the annual mean levels of all the parameters measured. Similarly, these trends were true in both sites during the dry season. Concentration levels during the wet season had little variations and generally lower than those of the dry season. During the dry season, the measured parameters exhibited greater degrees of decreases than those of the wet season.

Of the anions in both sites throughout the study period, Cl- had the highest 2- - mean concentration level, followed by SO4 , and then NO3 with the lowest. Of the + + cations, NH4 mean concentration was highest at the urban site. Na , the highest cation level at the rural site, was nearly similar to the urban level. Levels of Ca2+, K+ 12 A. Viernes and M. Laciste

MM LB 90

75

YMM = -1.059x + 35.92 60

YLB = -1.268x + 22.08 45 , umol/L 2- 4 30 SO 15

0 90

75

60

YMM = -0.702x + 26.10 45 , umol/L - 3 YLB = -2.526x + 27.04

NO 30

15

0 90

75 YMM = -2.376x + 46.17 60

YLB = -2.918x + 54.27 45 , umol/L -

Cl 30

15

0 2001 2002 2003 2004 2005 2006 2007 2008 2009 Year 2- 3- - Figure 5. Annual mean trends of anions (SO4 , NO , Cl ) of rainwater in Metro Manila and Los Baños from 2001 to 2009. Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 13

MM LB MM LB 150 150

125 125

100 100

75 75 , umol/ L , umol/ L 50 50 + + 4

25 Na 25 N H 0 0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2001 2002 2003 2004 2005 2006 2007 2008 2009

150 150

125 125

100 100

75 75 , umol/ L , umol/ L 2+ +

K 50 50 Ca 25 25 0 0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2001 2002 2003 2004 2005 2006 2007 2008 2009

150

125

100

, umol/ L 75 2+ 50 Mg 25 0 2001 2002 2003 2004 2005 2006 2007 2008 2009

+ + + 2+ 2+ Figure 6. Annual mean trends of cations (NH4 , Na , K , Ca , and Mg ) of rainwater in Metro Manila and Los Baños from 2001 to 2009. 14 A. Viernes and M. Laciste

MM LB

9 8 YMM =-0.200x+4.388 YLB =-0.171x+2.554 7 6

5 4

EC, mS/m 3 2

1 0 2001 2002 2003 2004 2005 2006 2007 2008 2009

9 8 YMM =-0.023x+5.951 YLB =-0.056x+6.001 7 6

5 4 pH value 3 2

1 0 2001 2002 2003 2004 2005 2006 2007 2008 2009

Year

Figure 7. Annual mean trends of EC and pH of rainwater in Metro Manila and Los Baños from 2001 to 2009. Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 15

Dry Season (DS) Wet Season (WS) METRO MANILA LOS BAÑOS 70 YDS = -2.8617x+52.185 YDS = -4.0491x+41.535 60 YWS = -0.0171x+25.359 YWS = 0.1525x+12.942 50

40 , umol/L 2- 4 30

SO 20

10

0

70 Y = -3.5358x+45.169 DS Y = -3.8598x+33.036 60 DS

50 YWS = 0.5407x+17.243 YWS = 0.5724x+8.2844 40

30 , umol/L - 3 20 NO 10

0

200 Y = -6.6742x+76.551 DS YDS = -12.349x+138.25 160 Y = -1.8043x+39.674 WS YWS = 0.3645x18.998 120

80 , umol/L -

Cl 40

0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2001 2002 2003 2004 2005 2006 2007 2008 2009

2- - - Figure 8. Seasonal trends (dry and wet seasons) of anions (SO4 , NO3 , Cl ) of rainwater in Metro Manila (left) and Los Baños (right) from 2001 to 2009. Dry Season (DS) Wet Season (WS) METRO MANILA LOS BAÑOS 350

300 YDS = -16.724x+188.31 YDS = -11.689x+109.55 250

200 Y = -1.1497x+50.378 WS Y = -0.8428x+27.34 , umol/ L WS + 150

100 N H 4 50 0 200

YDS = -6.2439x+70.917 Y = -7.9335x+103.04 160 DS

YWS = -6.3199x+66.308 Y = -1.0496x+24.494 120 WS , umol/ L

+ 80 Na 40

0 200 Y = -8.8144x+91.283 DS YDS = -4.5248x+36.774 160 Y = 0.1185x+6.1189 WS YWS = 0.328x+3.0784 120

, umol/ L 80 + K 40

0

100 Y = -3.4325x+32 YDS = -1.5748x+46.502 DS 80 Y = -3.2387x+28.092 YWS = -2.7065x+30.154 WS 60

, umol/ L 40 2+

Ca 20

0 100

80

YDS = -4.3692x+39.319 YDS = -2.3428x+22.948 60 , umol/ L Y = -0.3533x+6.5971 Y = -0.264x+5.5206

2+ WS WS 40 Mg 20

0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2001 2002 2003 2004 2005 2006 2007 2008 2009 Figure 9. Seasonal trends (dry and wet seasons) of cations (NH4+, Na+, K+, Ca2+, Mg2+) of rainwater in Metro Manila (left) and Los Baños (right) from 2001 to 2009. Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 17

Dry Season (DS) Wet Season (DS)

METRO MANILA LOS BAÑOS

YDS =-0.2335x+6.8896

Y =-0.4925x+4.8722 YWS =-0.0394x+2.3489 DS

YWS =-0.0251x+1.4919 Ec, mS/m

Y =-0.137x+6.775 YDS =-0.154x+7.106 DS

YWS =-0.052x+5.257 YWS =-0.024x+5.688 p H value

2001 2002 2003 2004 2005 2006 2007 2008 2009 2001 2002 2003 2004 2005 2006 2007 2008 2009 Year Year Figure 10. Seasonal trends (dry and wet seasons) of EC and pH in Metro Manila (left) and Los Baños (right) from 2001 to 2009. 18 A. Viernes and M. Laciste and Mg2+ follow, respectively, for both sites. Generally, concentration levels were higher at the urban site than those at the rural site.

Conclusion

Within the nine-year period of study, it was observed at both sites that the 2- - - + + + 2+ 2+ annual mean values of pH, EC, SO4 , NO3 , Cl , NH4 , Na , K , Ca , and Mg all follow a decreasing trend. The rate of decrease of anions concentrations in Metro Manila was lower than that of Los Baños, especially for nitrate ion. This means that for both sites, an improvement in air quality with respect to decreasing acidic emissions from mobile and industrial sources was observed; however, the rate was faster in Los Baños. Moreover, the rate of decrease of cations concentrations was greater in Metro Manila, except for calcium. The mean pH values throughout the study period were 5.72 at Los Baños site and 5.84 at Metro Manila site. This signifies that pH of rainwater in both the rural and urban sites was higher than the 5.6 value, which is considered acidic rainwater.

Recommendations

Increasing population and economic growth coupled with growing affluence will continue resulting in greater consumption of resources and energy through mass production for mass consumption. Consequently, acid deposition will continue to be one of the regional environmental issues and global issues as well. Continuous acid deposition monitoring to include additional monitoring sites throughout the Philippines is thus recommended to obtain more data that will give results which are more representative of the status of acid deposition in the Philippines. Future studies should also consider the number of vehicles plying the study areas for possible contribution of vehicular emissions to acid deposition, as well as contributions from industrial sources. It is also recommended that correlation of levels of wet deposition parameters with inland aquatic levels be studied.

Acknowledgment

The authors are thankful to the continuing support of EMB Central Office (CO) to the national EANET endeavors. Special acknowledgements are hereby given to the Research and Development Division of EMB-CO for their generous support and cooperation. Likewise, the authors extend their appreciation for Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites 19 the invaluable comments and suggestions of Ms. Ella S. Deocadiz, Ms. Lerma L. Dimayuga, Ms. Perseveranda-Fe J. Otico and Ms. Ma. Fatima Anneglo R. Molina.

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Trace metal speciation by sequential extraction in marine sediments of Calancan Bay, Sta. Cruz, Marinduque, Philippines

Dahlia C. Apodaca, Ph.D. Charo T. Balgua-Ocampo Senior Science Research Specialist Chemist III Chemistry Laboratory Services Section Mines and Geosciences Bureau Gina Flor C. Frankera-Resubal North Avenue Diliman, Quezon City Science Research Specialist II Email address: [email protected] Edita M. Macalalad Supervising Science Research Specialist

Heavy metal contamination is one of the many serious environmental problems affecting the society. Determining only the total metal concentration in sediments is not enough to gauge the potential impact of some heavy metals to aquatic organisms and human beings. To determine what chemical forms of the metals are bioavailable and have the potential for toxic effects, metal speciation is recommended. Through metal speciation, ways to abate the impact of heavy metals to the immediate community could be designed effectively.

This study on trace metal speciation was conducted in the municipalities of Sta. Cruz and Torrijos, province of Marinduque, particularly in Calancan Bay which used to be a repository of mine tailings generated from copper mine operations. The distribution of Copper (Cu), Lead (Pb), Arsenic (As), and Chromium (Cr) in the sediments collected from the Calancan Bay, Sta. Cruz, Marinduque,Philippines was determined by employing Tessier et al. speciation scheme to assess the geochemical mobility as well as the potential bioavailability of these heavy metals.

Keywords: sequential extraction, bioavailability, marine sediments, metal ions 22 D. Apodaca et al.

This study established current metal levels in the sediments collected from Calancan Bay, Marinduque; spatial variability in metal concentrations within the study area; and bioavailability of metals in sediments of Calancan Bay using the sequential extraction technique. The implications of the geochemical mobility of the heavy metals found in the sediments of Calancan Bay were also discussed and correlated to provide a better understanding of the environmental impacts of the presence of the tailings causeway in the bay. Copper present in sediment samples was found mostly in the organic fraction, while Pb was found to associate with the Fe/Mn oxides and hydroxides. As and Cr, on the other hand, were found in the lithogenic fraction of the sediment.

From 1969 to 1996, Marcopper Mining Corporation managed the large-scale, open-pit copper mining operations in Marinduque, Philippines. From 1975 to 1991, it resorted to near-shore marine disposal from its Tapian pit operations and made Calancan Bay, located on the northern part of Marinduque, its discharge point. Sixteen years of tailings disposal in Calancan Bay resulted in the formation of an approximately 5-km causeway with an estimate total area of 84 ha (Plumlee et al. 2000). It has become the permanent dwelling place of some Marinduqueños whose main livelihood is fishing. The corals, seaweed, and seagrasses at the bottom of the wide, shallow bay that used to serve as refuge for sea creatures are now covered with some 200 to 300 million tons of tailings. Due to the occurrence of several mining operations aside from the Marcopper operations, Marinduque has been plagued with mining-related environmental problems.

Due to foreseen potential impacts of these mine tailings, the Department of Environment and Natural Resources (DENR) created the Calancan Bay Rehabilitation Program (CBRP) on June 1989 to assess the environmental conditions of the bay attendant to the mining operations; and rehabilitate the bay to counterbalance, minimize and mitigate the impacts of the continuing disturbances on the coastal resources. In March 1997, the CBRP, in its final report, SEAMEO (1997) cited that it was not able to introduce effective measures to lessen the impact of heavy metal contamination in Calancan Bay. This is because it did not include in its report the means to establish the chemical forms of toxic metals present in the Calancan Bay. The concentration of the metals in the area may have exceeded some standards but the true impacts of the presence of these heavy metals have not been given much importance. Thus, over the years, the tailings may have posed some threat to the health of locals who constantly feed on fishes and marine life collected from the bay. A report of the Department of Health (DOH-UP 1997) indicated higher than acceptable lead levels in children residing near Calancan Bay. Trace metal speciation in sediments 23

With the vast contribution of mining operations to Philippine economy, it is therefore imperative to conduct a general assessment and monitoring of the environment in Marinduque prior, during, and post-mining operations. In this way, the magnitude of the environmental impact of near-shore disposal, which was practiced by Marcopper, will be assessed. In particular, processes that could affect the mobility or transport of metals in the marine environment may be identified. Metals can be found in more than one chemical form. The role and fate of toxic metals in the environment can result in several catastrophic events (e.g., mercury poisoning in Japan and mine-waste spill in the Philippines). The tendency and rate by which a metal participates in a geochemical or biological process depends on the physicochemical forms in which the metal occurs. Each physicochemical form of an element “speciation” can exhibit different bioaccumulation trends, toxicity limits, and effects on the degree of adsorption and desorption on the surfaces of suspended particulates, rates of transfer to the sediments, and overall transport in a seawater system. Thus, chemical speciation is one of the most important parameters that can enable humans to understand the mechanism by which pollutants act (Sadiq 1992). The identification of such classes of species can have huge importance in understanding the behavior and effects of heavy metals in natural and polluted areas both in the aquatic or terrestrial ecosystem.

Interest in the measurement of metal speciation began in the late 1960s following the mercury pollution incident in Minamata Bay in Japan. Laboratory studies revealed the differing toxicities of methylated and inorganic mercury species and the possibility of chemical and biological transformations from one form to another. The toxicity and fate of metal contaminants depend on the chemical forms and the quantification of these forms would be more meaningful than measuring the total metal concentrations. The principal purpose of measuring metal species is to determine the relative toxicity to aquatic biota and further understand metal interactions in the aquatic system, and their concentrations and transformations within and at the surface of organisms (Leppard 1983).

Thus, this study was conducted to: 1) establish the current metal levels in the sediments of the Calancan Bay; 2) evaluate the spatial extent of metal contamination away from the causeway; 3) investigate the changes in the metal concentrations in sediments of Calancan Bay relative to near-shore disposal of mine tailings through core drilling of sediments; and 4) perform chemical speciation of metals.

Review of literature

Results of the preliminary survey of marine contamination from mining-related activities on Marinduque Island, Philippines conducted by Plumlee et al. (2000) from 24 D. Apodaca et al.

October 14-19, 2000 showed that previous mining activities had contributed to the metal contamination in areas near the tailings causeway. As reported, high toxicity and elevated concentrations of Cu and other metals were measured in the pore water from two stations in the vicinity of the causeway. This observation was attributed to the presence of the causeway which is a constant source of Cu contamination in the surrounding environment. The group concluded that a comprehensive study must be conducted to establish the extent of contamination in the causeway within the Calancan Bay.

In view thereof, the geochemical properties of sediment are relevant in understanding the mobilization of heavy metals as well as the potential risks that these metals pose. This is usually done by determining the distribution and speciation of heavy metals in sediments. Chemical speciation can be defined as the process of identification and quantification of different species, forms, or phases of chemical present in a material (Niji et al. 2010). These chemical species are operationally defined according to the reagents used in their extraction. Selective sequential extraction offers information critical to the identification of the main binding sites, the strength of metal binding to the particulates, and the phase associations of trace elements in sediment. Results of sequential extraction determine the form of metal in sediments, which in turn, is highly significant in establishing the responses of the biological communities. These can then be translated to some ecotoxicological risk criteria. Selective sequential extraction gives a more realistic estimate of actual environmental impact of metals in the environment.

Tessier et al. (1979) first reported a scheme to sequentially extract metals from environmental samples. Since then, several schemes were reported integrating some modifications from the Tessier et al.'s speciation scheme. The Commission of the European Communities Bureau of Reference (BCR) provides a modified sequential extraction based on the schemes of Salomons and Forstner (1984), Towner (1984), Chester et al. (1985), Kersten and Forstner (1986), and Campanella et al. (1995). Ngiam et al. (2001) have noted that the BCR scheme ignores the carbonates while those procedures modified by Chester et al. (1985) and Campanella et al. (1995) introduced a humic associated fraction into the scheme.

In another study conducted by Medici et al. (2010), they used some parameters adopted from previous studies (Hakanson 1980; Ikem et al. 2003) to describe the contamination of metals in the river basin. These parameters include: Contamination Factor (CF), Degree of Contamination (Cd), Enrichment Index (EI), Individual Contamination Factor (ICF), and Global Contamination Factor (GCF). The results of sequential extractions were used to the ICF and the GCF. The ICF can be obtained by dividing the sum of the four extractions by the residual fraction of each sample sediment while the GCF for each site can be calculated by getting the Trace metal speciation in sediments 25 summation of the ICF of all the trace elements measured for a sediment sample (Ikem et al. 2003).

Yuan et al. (2004) analyzed 24 sediment samples from the East China Sea using the BCR sequential extraction method. They established high correlations between iron, cobalt, and nickel concentrations with the total organic carbon (TOC). They also found out that the concentration of elements in the acid soluble/reducible fractions were higher in the top sediments than that in the deeper positions implying direct relationship of distribution variation and depth. In a separate study by Ramirez et al. (2005), speciation of some heavy metals in sediments was performed to determine the influence of the El Salvador mine tailings. Among the notable observations include correlation of the concentration of the metals with the sediment grain size suggesting that metals preferentially associate with fine fractions. Moreover, results showed that the copper in the area was associated with mine tailings and was bioavailable.

Hung et al. (2009) determined the distribution and speciation of arsenic (As) and mercury (Hg) in sediments along the Gaoping Estuary-Canyon system in Taiwan. They reported that As and Hg are particle-reactive metals. Further, it was found out that most As is associated with Fe-Mn oxides while Hg is mainly associated with organic matter in mobile phases.

Cuong et al. (2006) have noted that studies on metal speciation in marine sediments have only been conducted in few countries including Turkey, Indonesia, Spain, and China. In fact, data on metal speciation in Singapore’s marine sediments were lacking. Results of their study showed that all metals measured, except cadmium (Cd), were more mobile and more bioavailable in Kranji than in Pulau Tekong. Cd had the highest mobility while chromium (Cr) was the least labile. Cd, Cu, Ni, and Zn have contrasting distributions in the marine sediments from both sites.

Thus, in consideration of the existing presence of mine tailings causeway along the Calancan Bay, Marinduque brought about by past mining activities, this study considered the use of selective sequential extraction method by Tessier et al. (1979) to assess the impact of the metals in marine sediments. Further, data on metal speciation in marine sediments, particularly in areas impacted by mining operations in the Philippines, are lacking to give a more reliable estimate of the bioavailability of these metals in the marine environment and consequently, establish the potential ecotoxicological risk posed by these metals to the biological community. 26 D. Apodaca et al.

Methodology

Sample collection

This study was conducted in the municipalities of Sta. Cruz and Torrijos, province of Marinduque in September 2003. Shallow sediments were collected from the Calancan Bay at near shore sampling points. Approximately, 2 kg wet weight, were collected at each near shore sampling point. Piston coring activity was carried out in October 2003, using the RPS Explorer (marine vessel of MGB) in the offshore areas of Calancan Bay along pre-determined sampling sites strategically derived from the bathymetry and topographic map of the study area (Figures 1 & 2 and Table 1). The said activity was facilitated with the use of a fabricated piston corer located at the starboard side of the vessel. A polyvinyl conduit (PVC) with varying length of 2-4 m was encased in the piston core. Weights were attached above the piston core, which acted as a triggering device for the piston core to penetrate the seabed once the safety lock is released. The piston core was positioned perpendicularly to the seawater surface to obtain maximum recovery of marine sediments. Splitting of piston core samples was carried out in the laboratory (PETROLAB Building) of the Mines and Geosciences Bureau in Quezon City. The core was split longitudinally and immediately logged. Logging was done megascopically and with the aid of standard color chart for soil and sediments. A sample log sheet was attached in the supporting information (Figure S1.) The collected sediment samples were then placed in polyethylene bags and stored in ice chests. The samples were refrigerated in the laboratory at 4°C prior to processing. Duplicate samples were obtained from each sampling station.

Also, during sample collection, in situ measurements of temperature, salinity, pH, turbidity, total dissolved solids (TDS), and dissolved oxygen (DO) were taken using a multi-parameter water quality probe (HORIBA). Condition and trends in water quality (temperature, TSS, turbidity, TDS, conductivity, salinity, and pH) were generally consistent across the study area. Metal analysis in coastal water was only limited to Cu, Pb and Cr due to the need to perform extraction using ammonium pyrrolidine dithiocarbamate in methyl isobutyl ketone solvent (APDC-MIBK). The extraction method was adopted from the Standard Methods for the Examination of Water and Wastewater published in 2005 and was validated by MGB Petrochemistry Laboratory unit. There were sites that were considered affected by point source discharges particularly with reference to metal levels. However, this particular study focused on determining the different chemical species of Cu, Pb and As in order to assess their mobility and relative toxicity in the marine sediments collected from the Calancan Bay. Trace metal speciation in sediments 27

Description of the test sites

the sediment samples were collected in different locations within the proximity of the causeway. Samples from trapichihan point were obtained in Sayao Bay while sediment samples from Calancan Bay were collected in four sampling stations, namely, Kawayan, Banot, Bathala, and panag. these four sampling sites were within the causeway. Both Bathala and panag were located on the left portion of the 5.0-km causeway while Banot and Bathala were situated nearest the causeway. Samples from torrijos were taken at the Salomague point and served as control samples, i.e., sediments collected from a pristine environment (unaffected by tailings). Shallow sediments were labeled as CB-02, CB-06, CB-09 (tip of the causeway), and CB-14 (Figure 2). Depth ranged from 8 to 12 m.

Figure 1. Location map showing Calancan Bay, Marinduque, Philippines. Source: Marinduque Travel Information at www.fnetravel.com 28 D. Apodaca et al.

Figure 2. Sampling sites of piston cored and shallow sediments from Calancan Bay, Marinduque. A total of nine core samples was gathered and individually logged. The marine sediments in the core samples predominantly consisted of fine to medium-grained coralline sand to mud/clay with coral/shell fragments. Dark greenish gray muddy sediment was observed above the mud/clay sediments in the core samples particularly in CB-10 and CB-13. The average length of the dark greenish gray muddy sediment was 40 cm. The observed dark greenish gray muddy sediments were believed to be the tailings disposed by the Marcopper Mining Corporation from the Mt. Tapian operation in the Calancan Bay area resulting in the formation of a causeway jutting into the said bay. Sites from which the core samples were taken are in Table 1 (Dagdag 2003). Trace metal speciation in sediments 29

Table 1. Description of sampling sites (piston core samples). Water Station Sample Latitude Longitude depth Remarks no. type (m) silty gray to light-gray sand CB-03 13°34.23’ 121°57.04’ 44.01 core with shell/coral fragments (1.90 m recovery) silty gray to light-gray sand CB-04 13°32.96’ 121°56.67’ 30.51 core with shell/coral fragments (0.1 m recovery) CB-05 13°33.42’ 121°57.23’ 36.21 core 0.3 m recovery light-gray medium to coarse grained sand with CB-08 13°34.30’ 121°58.05’ 42.02 core shell/coral fragments (0.3 m recovery)

CB-08A 13°34.19’ 121°57.94’ 39.78 core -do-

possible tailings deposit CB-10 13°33.70’ 121°58.99’ 45.18 core noted (3.7 m recovery) CB-12 13°34.27’ 121°59.09’ 57.05 core 1.9 m recovery light-gray mud (1.4 m CB-12A 13° 34.12’ 121°58.93’ 55.53 core recovery) possible tailings deposit CB-13 13° 33.11’ 121°58.99’ 41.28 core noted (2.75 m recovery) Note: CB-02, CB-06, CB-09 and CB-14 were shallow sediment samples and hence not subjected to core logging.

No sediment samples were collected in sampling points CB-01, CB-07 and CB-11 as these sampling areas were either shallow for the marine vessel to dock or blocked by several rock formations.

Reagents

Deionized water was obtained from Barnstead Thermolyne instrument (18 MΩ). Analytical grade nitric acid, hydrofluoric acid, acetic acid, hydroxylamine hydrochloride, hydrogen peroxide, and ammonium nitrate were used for extraction and wet digestion. Calibration solutions were prepared in deionized water from stock solutions (Merck). 30 D. Apodaca et al.

Figure S1. Log sheet for sediment sample collected via piston core at CB-03 site.

Determination of total metal content

All core and shallow sediment samples were digested with HF-HCl-HNO3 mixture for subsequent determination of total metal content using the Shimadzu 6650 Atomic Absorption Spectrophotometer (AAS). The technique presented herein has been validated by the MGB Chemical Laboratory Services Section and that of the Petrochemistry unit, PETROLAB. The technique has been used by MGB for total metal analysis whenever the matrix involves sediments, rocks and soils. The technique is an in-house (MGB) validated procedure way back in the 80s during the implementation of the United Nations Development Programme (UNDP) at MGB. Metal concentrations were determined for Cu, Pb, Cd and Cr. Concentrated HCl (10 mL), concentrated HNO3 (5 mL) and concentrated HF (2 mL) were added to 1.0 g of dry sediment in a Teflon beaker. After the effervescence has disappeared, the beaker was heated on a hotplate at about 100oC (on top of sandbath to prevent the solution from boiling). The acid solution was then evaporated to near dryness. Then,

5 mL of HNO3 was added and the beaker was again heated and the solution was evaporated to near dryness. The addition of 5 mL concentrated HNO3 accompanied Trace metal speciation in sediments 31 with heating to near dryness was performed two times. After which, the solution was diluted with water and was heated to boiling. The decomposed solution was filtered with #2 Whatman filter paper and the filtrate was collected in a 100-ml volumetric flask. The solution was diluted with water to make a 100 mL solution before metal analysis was performed using Flame Atomic Absorption Spectroscopy (FAAS). Total mercury content of all samples was also determined using the Automatic Mercury Analyzer (AMA-254) which requires no sample decomposition. The analysis of arsenic was performed using the Hydride Generation (VGA)-AAS method using sodium borohydride as reductant.

Speciation analysis

To determine the metal speciation or element distribution in sediments, Tessier's sequential extraction procedure was employed (Scheme 1). Each sample was dried in the oven at 40oC and was analyzed on as received basis. Extraction of the samples was done using 50-ml polypropylene centrifuge tubes to minimize losses of solid materials. Standard reference materials were also subjected to sequential extraction to test the accuracy of the method. GXR-2, JSD-1, and GSD- 2 are commercially available whereas Asiga and Tumbaga are in-house reference materials at MGB.

Results and discussion

Total metal concentration

Table 2 summarizes the total concentration of Cu, Pb, Cd, Hg, Cr and As in shallow sediment samples collected along the Calancan Bay. As expected, the results indicated high Cu concentration and low to moderate metal enrichment with respect to the occurrence of Pb, As, Cr, and Hg. The metal concentrations measured characterized an area that is highly mineralized with Cu deposits and other associated elements as manifested in Figure 3.

Levels of metals in sediments collected along the Calancan Bay were compared with the metal levels in sediments collected from Salomague Point in Torrijos, located north of Boac. It can be gleaned from Table 2 that the metal levels in samples obtained in Torrijos were relatively lower compared with metals present in sediments collected in Calancan Bay. This shows the effects of anthropogenic activities that have been ongoing in Calancan Bay. 32 D. Apodaca et al.

Scheme 1. Flow chart of the sequential extraction procedure adopted from Tessier at al. Trace metal speciation in sediments 33 2 1 Cd <1 <1 <1 <1 <1 <1 <1 <1 <1 4 7 8 20 22 20 21 54 2.5 Hg* 10.5 N.A. 6 9 Cr 14 37 31 19 22 37 33 37 <1 7 9 3 3 2 3 5 2 4 2 3 As Total metal content (μg/g dry wt) 3 4 4 5 9 8 78 13 46 Pb <1 <1 79 53 20 12 54 69 Cu 915 841 1281 1337 3589 57' 50" E 56' 38" E 58' 11" E 58' 26" E 57' 59" E 59' 9" E 06' 17" E 06' 25" E o o o o o o o o 33' 45" N 33' 31" N; 31' 15" N; 32' 27" N; 33' 46" N 32' 11" N 19' 45" N; 19' 51" N; o o o o o o o o Coordinates 13 13 13 13 121 121 121 122 122 121 121 13 13 13 13 121

Sampling points T ailings end of causeway (CB-09) T ailings Bathala Control 1 – T orrijos Control 2 – SW T orrijos T ailings with seawater (CB-09) Station 14 (depth: 10 m, CB-14) Station #6 (depth: 12 m, CB-06) Station #2 (depth: 8 m, CB-02) Station 14 Sed 2 (depth: 12 m, CB-14) Station #9 (depth: 8 m) Station #6 Bottom (CB-06) Table 2. Total metal content in shallow sediment samples. *parts per billion (ppb) 34 D. Apodaca et al.

Figure 3. Total metal content (Cu, Pb, Cr, Cd and As) measured from various sampling locations within the Calancan Bay, Marinduque.

Heavy metal content of tailings

Tailing samples were analyzed for its Cu, Pb, As, Cr, Hg, and Cd content. The metals considered in this study were monitored to determine if the levels were within the regulated limits stated in DENR-DAO 35. It was established that the tailings still contain incredibly high concentration of Cu and low to moderately high concentrations of As, Cd, Cr, Pb, and Hg. Results obtained from the analysis were compared with those that were presented in the report of the independent United States Geological Survey (USGS) team in 2004. However, such comparison is limited only to the six heavy metals analyzed in this study and that the USGS report made no mention of whether the reported values were based on dry weight or as received basis (Table 3). In this study, it is worth noting that the tailings contain significant concentrations of Pb. With respect to Hg, no comparison can be made since the independent USGS team did not collect samples to be analyzed for Hg content. Comparison was made only to ascertain if there has been considerable changes on the concentration levels of these heavy metals in Calancan Bay from the time the USGS reported the data until the study was conducted. The authors acknowledged that there will be deviations between these data.

Spatial extent of the metal enrichment in Calancan Bay

This study also determined and investigated the spatial extent of metal contamination in sediments of the Calancan Bay, which has become the dumping Trace metal speciation in sediments 35

Table 3. Comparison of the total metal content obtained in this study with that of the 2004 report of the independent USGS team. Concentrations are expressed in parts per million. This study USGS 2004 Report Tailings end of Tailings with sea- Parameter Calancan tailings, causeway water Sand fraction (-120 mesh) (-120 mesh) Longitude 121o58’11” 121o58’11” 121o57'9" Latitude 13o33’45” 13o33’45” 13o33'2" Arsenic (As) 2 2 <15 Cadmium (Cd) 2 1 4.4 Chromium (Cr) 31 33 <25 Copper (Cu) 1281 3589 961 Lead (Pb) 78 46 10 Mercury (Hg) Not analyzed 54* Not determined *parts per billion (ppb) ground of tailings from the Tapian pit operations of Marcopper. Samples collected from a distance of about less than 1 km from the end of the causeway, yielded an average of 878 ppm Cu, 8 ppm Pb, 4 ppm As, 37 ppm Cr, 14 ppb Hg and <1 ppm Cd. High concentrations of metals at some points far from the causeway can be attributed to the continuous erosion (apparently due to water current) of the tailings dumped in the Calancan Bay. The components of the tailings had slowly dispersed away from the causeway. It was observed that a portion of the causeway was already detached from the whole 5-km stretch of the tailings mass.

To investigate the possibility of the contaminants being slowly dispersed into the immediate environment of the bay and that the bay contains voluminous loads of tailings deposited at the bottom, the Marine Geological Survey Division of MGB had collected core samples from several points within 2.5 to 4.4 km far from the end of the causeway. Total metal concentration of the core samples indicated elevated metal levels between those distances and between specified depths as shown in Figures 3a to 3c. From the results of the trace metal analysis of the core samples, going from the topmost portion of the core to the bottom, the total metal concentration generally decreased. This trend clearly supported the observation stated in Dagdag’s unpublished report in 2003 that the observed dark greenish gray muddy sediments could be those tailings disposed by the Marcopper Mining Corporation from the Mt. Tapian operations. On the other hand, even if there was no clear trend as to the decrease in the metal level from top to bottom of some core samples, results 36 D. Apodaca et al.

Figure 3a. Spatial and vertical distribution of Cu (ug/g sediment) in marine sediments of Calancan Bay.

Figure 3b. Spatial and vertical distribution of As (ug/g sediment) in marine sediments of Calancan Bay. Trace metal speciation in sediments 37

Figure 3c. Spatial and vertical distribution of Pb (ug/g sediment) in marine sediments of Calancan Bay. still showed uniformly high concentrations of metals found in sediments collected on the topmost portion of the core samples. This suggested possible contributions from human and mining activities. However, it was notable too, that at a certain depth, based on qualitative characterization of sediments obtained via drilling (9 core samples, Table 1), drilled samples were found to contain only what appeared to be mine tailings.

In terms of Cr, Cd, and Hg metal levels, results showed that these metals were dispersed uniformly within the given area and therefore may be considered as part of the geological make-up of the area. However, for Cu, Pb, and As, wide variations in the metal concentrations may denote anthropogenic inputs.

Correlation studies

Statistical treatment of the data, i.e. data on the total metal level of Cu, Pb, Cd, Hg, Cr and As, indicated quite strong correlation of As with Cd and Cr as well as between Cu and Pb (positive values). These observations denote that the six heavy metals measured may be classified into two groups: 1st group with As, Cd, and Cr, and the 2nd group with Cu and Pb (Table 4). 38 D. Apodaca et al.

Table 4. Correlation table of the Total Metal level of Cu, Pb, Cd, Hg, Cr and As in sediment samples.

Copper Lead Arsenic Chromium Mercury Cadmium Cu 1 Pb 0.45521 1 As -0.0789 0.332311 1 Cr 0.23413 0.28217 0.0948 1 Hg 0.28787 0.291901 0.044961 -0.09295 1 Cd -0.6337 0.384357 0.717637 0.592312 -0.39581 1 Correlation was performed to look into the possible association or groupings of metals at the different fractions. Table 4 illustrates strong correlation between Cu and Pb (r = 0.94658) in fraction 1 or the exchangeable fraction which implies that these two metals associate with one another in their most mobile and bioavailable form. However, it should be noted that as the two above mentioned metals were further fractionated, the close association between the two metals tends to decrease.

A moderately high correlation between Cr and As was observed in fraction 1 (exchangeable) (r = 0. 4535) and fraction 3 (Fe/Mn oxides and hydroxides) (r = 4728). Poor correlation among metals in fraction 5 (lithogenic fraction) was also observed. Calculated correlation values are <1 (negative values).

Speciation analysis (Metal distribution)

The chemical forms of various metals in the environment dictate their potential for toxic effects to human beings, aquatic organisms, etc. Data gathered from speciation studies may be utilized to create a general mining environmental and health assessment of the Calancan Bay. Results of speciation experiments revealed an already obvious fact that metals are heterogeneous with respect to their geochemical mobility. Associations of the metals can be subdivided into five fractions (Pickering 1986) and these subdivisions were also employed in the separation of metals as reported by Tessier et al. (1979). Fraction 1: Water-soluble, weakly adsorbed and exchangeable fractions Fraction 2: Bound to carbonates Fraction 3: Bound to hydrous oxides of Fe, Mn Fraction 4: Bound to organic matter Fraction 5: Inert fraction (detrital lithogenic minerals)

Considerable attention was given to metals in Fractions 1 and 4 as these forms of metals pose hazard to organisms. Metals that are weakly adsorbed and Trace metal speciation in sediments 39 exchangeable are considered mobile, and solubilize easily in aqueous systems. On the other hand, metals that have been found to associate with organic matter have the potential to bioaccumulate and enter the food web. Bioaccumulation is a process facilitated by aquatic organisms in which as these metals are introduced to the food web, their concentrations tend to increase as they go up the food chain. Metals form complexes with carbonates and hydrous oxides of Fe and Mn and can be released into the porewater if there will be considerable change in the pH. Also, the release of metals in Fe-Mn fractions may take place under reducing conditions. Inert fractions are usually comprised of metals making up the geologic make up of a specific area/ location (Marin et al. 1997).

In this speciation study, only Cu, Pb, Cr, and As metals were subjected to partitioning. Cd was not included because its occurrence in the sediment samples was below the detection limit of 1 ppm. In the case of Hg, loss may actually be incurred during the 3rd and 4th stages of the speciation scheme since at these stages, temperature setting was quite high for Hg and thus would result in the volatilization of some of the organic forms of Hg (boiling point of methylmercury is around 85oC).

As previously reported (Medici et al. 2011), the Individual Contamination Factor (ICF) manifests the risk of contamination of a site by a metal pollutant. On the other hand, the Global Contaminant Factor (GCF) illustrates the potential risk to basin water contamination by basin sediments. Results of the speciation study performed on the marine sediments from Calancan Bay showed very high ICF (values ranging between 3 and 12) for Cu measured in areas from several locations: Station #6, CB- 12A, CB-13, CB-10, CB-03, CB-08, CB-08A, and Mogpog. Very high ICF factor as high as 44 was also established for Pb at station CB-12A-06 (core sample, depth: 127-173 cm). In the case of As, ICF factors of 4.4 – 5.1 was achieved for core samples collected from stations CB-08 (top) and CB-08A (depths: 0-35 cm and 70- 115 cm). Low ICF factor was obtained for Cr such as 1.30 for Sta. 14, 10m depth and SW-2 Torrijos. With these observed metal enrichment, GCF factors calculated were generally high in almost all sampling stations within the bay.

Validation of the speciation scheme

To check the suitability of the Tessier et al.'s method in fractioning the heavy metals found in marine sediments and mine tailings, standard reference materials were also subjected to speciation studies. In this study, Asiga and Tumbaga, in-house reference materials at MGB; GXR-2, a soil (0-18 cm depth) from Park City, Utah; and JSD-1, obtained from the Japan Geological Survey were utilized as standard reference materials. Recovery of metals present in these reference materials is presented in Tables 5a to 5c. 40 D. Apodaca et al.

Table 5a. Copper (Cu) speciation data of some standard reference materials. Cu, ppm Standard Sum of Total Percent reference Frac- Frac- Frac- Frac- Frac- F1 to metal recovery material tion 1 tion 2 tion 3 tion 4 tion 5 F5 content GXR-2 2.11 4.16 7.11 22.85 42.99 79.22 74 107.1 JSD-1 2.66 0.97 3 1.3 13 20.93 22 95.1 Asiga 8 2 18 2 171 201 200 100.5 Tumbaga 0.29 2.44 14 26 53 95.73 96 99.7

Table 5b. Lead (Pb) speciation data of some standard reference materials. Pb, ppm Percent Standard recovery reference Frac- Frac- Frac- Frac- Frac- Sum of Total material tion 1 tion 2 tion 3 tion 4 tion 5 F1 to metal F5 content GXR-2 6.2 147.3 394.2 22.3 102.6 672.6 705.2 95.4 JSD-1 1.28 0.11 6.06 1.21 3.69 12.35 12.90 95.7 GSD-2 5.25 1.58 14.38 0.99 9.51 31.71 31 102.3 Tumbaga <0.05 <0.05 4.4 0.75 16.5 21.65 16 135.3

Table 5c. Arsenic (As) speciation data of some standard reference materials. As, ppm Percent Standard recovery reference Frac- Frac- Frac- Frac- Frac- Sum of Total material tion 1 tion 2 tion 3 tion 4 tion 5 F1 to metal F5 content GXR-2 0.29 0.67 2.20 0.40 18.4 21.96 31 70.8 JSD-1 0.30 <0.05 <0.05 0.10 1.8 2.2 2.42 90.9 Asiga 0.27 0.07 0.08 0.12 8.3 8.84 10 88.4 Tumbaga 0.23 0.15 <0.05 1.1 2.0 3.48 2 174 GSD-2 0.27 0.97 <0.05 <0.05 2.6 3.88 6.4 60.6 Trace metal speciation in sediments 41

The recovery of the sequential extraction was calculated using equation 1: Recovery = [(Cfraction 1 + Cfraction 2 + Cfraction 3 + Cfraction 4 + Cfraction 5) / Ctotal ] x 100 (1)

The Tessier et al.'s method yielded very good recoveries for the fractionation of Cu and fairly good recoveries for the three metals speciated. The method used is reliable and repeatable for the speciation of metals in marine sediments.

Copper

The calculated percentage of potentially available copper was obtained by adding the percent-extracted metal in the four fractions (fractions 1-5). The amount of potentially available copper across all sediment samples (shallow and core) yielded an average of 58.5%. As seen from the distribution plot in Figure 4a, Cu present in shallow sediment samples was found mostly in the organic fraction which implies that Cu has the potential to form complexes with organic matter such as humic substances, phytoplanktons, amino acids, etc. This suggests that Cu tends to bioaccumulate in the long run.

Core samples were also subjected to speciation. However, during the splitting of samples, it was found out that only one sample yielded complete (good) recovery. For this study, comparison cannot be made among the core samples obtained and thus, the discussion on speciation results would focus on the CB-12A core sample only. Vertical distribution of Cu particularly at point CB-12A indicates significantly high concentration of Cu, which was also found in the organic fraction (fraction 4). Furthermore, a uniform concentration of Cu was obtained within a thickness of 183 cm core sample. Percent extractability for Cu in the organic fraction in this core sample is approximately 70%.

Lead

Lead was found to associate with the Fe/Mn oxide and hydroxide fraction and that the average percent potentially available across all samples (shallow and core) is around 70.3%. This puts forward the possibility that the Pb in the sediments exists in the form of oxides. Possible mechanisms of reaction involving Pb in the marine environment are given in equations 2 and 3: SOH + Me SOMe + H+ (2) + 2SOH + Me S2O2Me + 2H (3)

where: Me stands for the Pb or the metal associated in this fraction and SOH are the surface hydroxyls in the sediment.

Speciation results for core sample CB-12A indicate no clear trend as to the amount of Pb that can be extracted in each fraction except in the case of fraction 1, 42 D. Apodaca et al.

Distribution Plot for Cu 100% 90% 80% 70% 60% 50% 40% 30% 20% % Extraction 10% 0% Sta. # 6 Sta. # 2 6 Bottom th: 12 m 14 Sed 2 Sta. 14 (depth: 8m) (depth: 12m) Seawater Causeway Tailings with Tailings (depth: 10m) Tailings End of Tailings #1 (depth: 8m) Sta. 2 Sediment Tailings Bathala Tailings

Sampling Points

Legend: Fraction 1 Fraction 3 Fraction 5 Fraction 2 Fraction 4 Figure 4a. Distribution of the different forms of Cu in the different sediment samples by fraction and per location site.

Distribution Plot for Pb 100% 90% 80% 70% 60% 50% 40% 30%

% Extraction 20% 10% 0% Sta. # 6 Sta. # 2 6 Bottom Sta. # 14 Seawater Causeway Tailings Tailings with SW-2 Torrijos SW-2 Tailings End of Tailings Control Torrijos Tailings Bathala Tailings

Sta. 2 Sediment #1 Sampling Points Legend: Fraction 1 Fraction 3 Fraction 5 Fraction 2 Fraction 4

Figure 4b. Distribution of the different forms of Pb in the different sediment samples by fraction and per location site. Trace metal speciation in sediments 43 wherein percent extractability was noted at 10%. An important indication that needs to be verified is the close association of Pb with Cu. When there is potentially available Cu (58.5%), there could also be a possibility that Pb would also be potentially available even at higher percentage, as revealed in the speciation experiments. The distribution plot shown in Figure 4b gives the different forms of Pb in the different sediment samples collected along Calancan Bay.

Arsenic

Evaluation of the speciation studies relative to all samples taken reveal that most of the As can be found in fraction 5 or the detrital lithogenic fraction which means that the As present in the sediment samples is not potentially available in as much as in Cu or Pb. Average percent availability is approximately 34.64%. Arsenic is likely incorporated in the aluminosilicate minerals and therefore may unlikely be released into the porewater by dissociation.

Vertical distribution of As, as shown in Figure 4c, indicates no clear trend as to the percent extractability of As in the lithogenic fraction.

Chromium

The distribution plot for Cr as displayed in Figure 4d clearly shows that Cr was partitioned between three fractions only and that majority of Cr was more distributed in fraction 5 or the detrital lithogenic fraction. Moreover, this result supported the claims that the Cr present in the sediment samples is of geogenic source due to the uniform spatial distribution of Cr within the sampling area. Average percent potential availability of Cr is only 19.46%.

With regards to the vertical distribution of Cr at CB-12A, quite uniform percent extractability (~85%) was obtained starting from 125 cm (midway the actual thickness of the core sample) going down to the bottom sample. Speciation results for both Cr and As clearly jibed with correlation findings that Cr, As, and Cd closely associate with one another indicating that the three metals may have geogenic origin. The tabulated speciation data are given in Tables 6 and 7 while the vertical distribution plots are given in Figure S2. 44 D. Apodaca et al.

Distribution Plot for As 100% 90% 80% 70% 60% 50% 40% 30% 20%

% Extraction 10% 0%

Sta. # 2 Sta. # 6 Sta. 14 6 Bottom

SW-2 Torrijos Tailings Bathala Control Torrijos

Sta. 2 Sediment #1 Tailings with Seawater Tailings End of Causeway Sampling Points Legend: Fraction 1 Fraction 3 Fraction 5 Fraction 2 Fraction 4 Figure 4c. Distribution of the different forms of As in the different sediment samples by fraction and per location site.

Distribution Plot for Cr 100% 90% 80% 70% 60% 50% 40% 30%

% Extraction 20% 10% 0%

Sta. 14 Sta. # 2 Sta. # 6 6 Bottom

SW-2 Torrijos Tailings Bathala Control Torrijos

Sta. 2 Sediment #1 Tailings with Seawater Tailings End of Causeway Sampling Locations Legend: Fraction 1 Fraction 3 Fraction 5 Fraction 2 Fraction 4

Figure 4d. Distribution of the different forms of Cr in the different sediment samples by fraction and per location site. Trace metal speciation in sediments 45 Cr μg/g dry wt 4.0 < 0.1 2.9 < 0.1 < 0.1 3.0 3.0 0.67 < 0.1 < 0.1 2.0 6.0 As μg/g dry wt < 0.1 < 0.1 0.26 0.40 0.64 < 0.1 0.34 0.42 0.14 0.13 0.30 0.34 Fraction 3 Pb μg/g dry wt 34 31.7 0.5 13 4.8 7.8 5.3 9.5 < 0.1 1 < 17 Cu μg/g dry wt 172 16.9 < 0.1 392 9.4 42.8 49.6 < 0.1 < 0.1 < 0.1 < 0.1 14.4 Cr μg/g dry wt < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 As μg/g dry wt < 0.1 0.17 < 0.1 < 0.1 0.28 0.29 0.29 0.22 0.23 0.37 < 0.1 0.53 Fraction 2 Pb μg/g dry wt 4 10.9 0.4 0.57 0.56 0.68 1.9 0.51 0.29 0.22 < 0.1 2 Cu μg/g dry wt 316.5 315.3 1.8 403 8.2 66.4 73.5 0.8 1 1 0.73 85.1 Cr μg/g dry wt < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 As μg/g dry wt 0.13 < 0.1 0.18 0.15 0.59 0.49 0.33 0.18 0.20 0.27 0.35 0.27 Fraction 1 Pb μg/g dry wt 3 < 0.1 0.4 0.3 < 0.1 < 0.1 < 0.1 0.58 0.55 0.73 0.51 0.33 Cu μg/g dry wt 243 8.8 0.1 6.7 0.62 8.6 5.4 0.5 0.7 0.8 0.11 3.7 - Concentrations of Copper, Lead, Arsenic and Chromium in Fractions 1-3 per sample site employing the speciation scheme by Tessier et al. Sample locations T ailings end of Causeway T ailings with Sea water Sta. 2 Sediment #1 Sta. 2 Sediment #1 (depth: 8 m) T ailings Bathala Sta. #2 (depth: 8 m) Sta .#6 (depth: 12 m) 6 Bottom Sta. 14 Sed 2 (depth: 12 m) Sta. 14 (depth: 10 m) Control T orrijos SW-2 T orrijos CB-12A-07 (173-183 cm) Table 6. 46 D. Apodaca et al. Cr μg/g dry wt 0.19 4.0 1.3 0.10 0.14 0.27 2.4 2.6 5.4 5.1 3.6 As μg/g dry wt < 0.1 0.31 < 0.1 0.11 0.23 < 0.1 0.42 0.67 4.1 4.4 0.38 Fraction 3 Pb μg/g dry wt 8 9 6 7 5.5 8.7 5.1 1.2 3.3 1.9 9.2 Cu μg/g dry wt 10 26 5 15 51.7 16.4 110 <0.1 2.6 2.7 43.2 Cr μg/g dry wt < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 2.2 2.24 11.9 As μg/g dry wt 0.19 < 0.1 < 0.1 < 0.1 0.31 < 0.1 0.38 0.79 0.42 0.40 0.25 Fraction 2 Pb μg/g dry wt 1.5 2 1 2 4.7 2.2 4.9 < 0.1 < 0.1 < 0.1 1.5 Cu μg/g dry wt 19.1 79 17 90 66.2 124.9 72 0.39 0.21 0.10 107 Cr μg/g dry wt < 0.1 < 0.1 < 0.1 <0.1 < 0.1 < 0.1 0.58 0.87 < 0.1 0.10 0.18 As μg/g dry wt 0.27 0.22 0.20 0.18 0.36 0.24 0.36 0.43 0.26 0.29 0.28 Fraction 1 Pb μg/g dry wt 0.51 0.55 0.55 0.11 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 Cu μg/g dry wt 2.2 6.5 2.8 5.5 5.4 4.6 3.6 0.35 0.14 < 0.1 4.3 Sample locations CB-12A-06 (127-173 cm) CB-12A-05 (117-125 cm) CB-12A-04 (26-110 cm) CB-12A-03(20-26 cm from bottom) CB-12A-02 (10-20 cm) CB-12A-01 (bottom: 10 cm) CB-13-02 (126-165 cm) CB-13-01 (0-126 cm) CB2-13-02 (50-115 cm) CB2-13-01 (0-50 cm) CB1-10-02 (100-147 cm) Table 6. Continuation Trace metal speciation in sediments 47 Cr μg/g dry wt 3.2 2.3 3.3 3.0 2.3 2.7 3.5 3.8 2.8 3.8 4.2 As μg/g dry wt 0.37 0.54 0.28 0.33 0.42 1.2 2.7 2.95 0.43 1.7 0.13 Fraction 3 Pb μg/g dry wt 4.46 6.1 5.5 7 3.3 2 2.9 5.8 11.2 12.1 10.1 Cu μg/g dry wt 33 27.6 43 16 9 4.6 < 0.1 5.5 0.64 17 34 Cr μg/g dry wt 0.88 0.80 0.42 < 0.1 1.1 < 0.1 4.5 2.2 < 0.1 0.68 1.1 As μg/g dry wt 0.47 < 0.1 0.26 0.43 0.44 0.69 0.38 0.28 < 0.1 0.57 0.44 Fraction 2 Pb μg/g dry wt 0.18 1.7 < 0.1 3 0.99 < 0.1 < 0.1 < 0.1 < 0.1 1.22 < 0.1 Cu μg/g dry wt 11 22.8 10 13 8 < 0.1 5 29 0.3 20.5 3.6 Cr μg/g dry wt 0.52 < 0.1 0.81 0.29 0.98 0.64 0.12 0.18 0.46 0.70 0.98 As μg/g dry wt 0.37 < 0.1 0.39 0.28 0.29 0.38 0.19 0.17 0.29 0.68 0.31 Fraction 1 Pb μg/g dry wt < 0.1 < 0.1 0.54 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 Cu μg/g dry wt 1 2.4 1 0.5 1.14 0.5 < 0.1 0.35 < 0.1 1 2.1 Sample locations CB1-10-01 (0-100 cm) CB-03-02 (30-59 cm) CB-03-01 (0-30 cm) CB-04-02 (10-15 cm) CB-04-01 (0-10 cm) CB2-10-02 (107-120 cm) CB2-10-01 (0-20 cm) CB-12-02 (147-177 cm) CB-12-01 (0-10 cm) CB-03-04 (164-188 cm) CB-03-03 (59-164 cm) Table 6. Continuation 48 D. Apodaca et al. Cr μg/g dry wt 1.6 1.1 5.3 4.0 0.62 2.9 2.8 < 0.1 As μg/g dry wt 0.14 0.21 0.38 0.29 < 0.1 < 0.1 0.15 < 0.1 Fraction 3 Pb μg/g dry wt 1.3 0.39 10.2 5.4 2.5 0.22 2.7 2 Cu μg/g dry wt 5 4 35.5 23 53.5 0.35 < 0.1 464 Cr μg/g dry wt 0.45 < 0.1 0.66 0.28 < 0.1 < 0.1 < 0.1 < 0.1 As μg/g dry wt 0.41 0.49 0.24 0.47 0.27 0.36 0.53 <0.1 Fraction 2 Pb μg/g dry wt 0.76 1.1 5.9 4 0.7 0.35 < 0.1 < 0.1 Cu μg/g dry wt 16 16 55.8 14 28 < 0.1 0.23 187 Cr μg/g dry wt 0.52 0.46 0.52 0.64 0.93 0.58 0.46 < 0.1 As μg/g dry wt 0.32 0.27 0.39 0.37 0.31 0.31 0.31 0.25 Fraction 1 Pb μg/g dry wt < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 0.22 Cu μg/g dry wt 1 1 3.1 1 1 0.4 < 0.1 5.2 Sample locations CB-05-02 (15-30 cm) CB-05-01 (0-15 cm) CB-08-02 (top) CB-08-01 (bottom) CB-08A-03 (70-115 cm) CB-08A-02 (35-70 cm) CB-08A-01 (0-35 cm) Mogpog Brgy. Malusak Table 6. Continuation Trace metal speciation in sediments 49 Cr μg/g dry wt 31.3 20.1 25.4 21.5 30.4 42.8 17.7 22.1 3.1 < 0.1 2.3 3.5 29.2 31.1 23.9 21.3 37.2 29.2 24.7 As μg/g dry wt 1.3 4. 0 4.7 1.7 2.7 2.4 0.92 1.5 1.5 1.7 4.2 1.8 2.7 3.0 3.4 3.7 3.0 1.3 4.2 Fraction 5 Pb μg/g dry wt 4.9 3.4 0.4 2.2 1.9 4.5 0.23 6.6 < 0.1 0.26 4.3 1.4 0.74 3.9 < 0.1 3.7 5.07 6.4 8.0 Cu μg/g dry wt 40.3 57.8 31.8 92.3 34.1 52 30.1 72.3 9.0 10.4 38 31.5 66.7 77.2 27.3 105.1 56.7 1053.3 207.4 Cr μg/g dry wt 2.6 1.4 1.5 0.52 < 0.1 < 0.1 < 0.1 < 0.1 2.0 0.81 3.0 2.5 1.2 1.2 2.4 < 0.1 3.0 1.4 2.5 As μg/g dry wt 0.24 < 0.1 0.2 < 0.1 0.17 0.24 0.33 < 0.1 0.2 0.27 0.41 < 0.1 0.21 0.29 0.58 0.28 2.6 < 0.1 0.14 Fraction 4 Pb μg/g dry wt 6.9 0.2 < 0.1 0.51 < 0.1 3.0 < 0.1 0.29 0.64 1.2 0.64 0.52 < 0.1 < 0.1 < 0.1 1.45 1.2 2.7 9.0 wt Cu μg/g dry 214.1 424.9 249.1 434 324.1 456 265 484 6.0 8.0 7.4 19 715.4 598.2 15.8 185 16 1842.6 286.4 Sample locations Concentrations of copper, lead, arsenic and chromium in Fractions 4 5 per sample site employing the speciation scheme by Tessier et al. CB-13-02 (126-165 cm) CB-12A-01 (bottom: 10 cm) CB-12A-02 (10-20 cm) CB-12A-03 (20-26 cm from bottom) CB-12A-04 (26-110 cm) CB-12A-05 (117-125 cm) CB-12A-06 (127-173 cm) CB-12A-07 (173-183 cm) SW-2 T orrijos Control T orrijos Sta. 14 (depth: 10 m) Sta. 14 Sed 2 (depth: 12 m) 6 Bottom Sta .#6 (depth: 12 m) Sta. #2 (depth: 8 m) T ailings Bathala Sta. 2 Sediment #1 (depth: 8 m) T ailings with seawater T ailings end of causeway Table 7. 50 D. Apodaca et al. Cr μg/g dry wt 21.3 11.2 12.2 28.4 16 32.3 25.3 33.6 72.1 20.3 23.6 25.6 19.1 26.9 18.9 16.3 13 12.5 12.8 28.8 13.4 17.3 As μg/g dry wt 9.9 0.22 0.17 2.3 4.0 3.7 1.7 1.2 3.13 40.6 3.6 3.3 134.1 3.23 3.4 0.72 3.15 129 2.0 38.9 4.6 3.6 Fraction 5 Pb μg/g dry wt 10.4 3.3 0.46 < 0.1 5.0 7.4 < 0.1 0.46 9.8 3.8 25.9 4.0 6.24 6.5 2.66 3.8 0.35 1.4 126.9 5.6 2.6 1.4 Cu μg/g dry wt 305 22.3 37 23.1 56 36.7 74 74 35 48.5 110 68.5 45 35.3 35 17.8 31.4 22.6 32.2 64.9 12.7 16.7 Cr μg/g dry wt 0.41 1.6 2.5 2.2 0.72 2.3 0.27 0.27 2.9 2.6 2.0 1.8 1.3 1.8 0.52 2.9 1.5 1.1 3.0 3.8 0.96 1.0 As μg/g dry wt 0.15 0.13 < 0.1 0.18 0.14 0.21 < 0.1 < 0.1 0.28 0.18 0.29 < 0.1 0.23 0.13 < 0.1 0.87 < 0.1 0.15 0.49 0.26 < 0.1 0.42 Fraction 4 Pb μg/g dry wt 0.64 < 0.1 1.62 4.0 1.7 < 0.1 0.69 1.2 0.87 1.5 < 0.1 < 0.1 0.12 1.4 < 0.1 3.9 0.58 < 0.1 2.8 < 0.1 < 0.1 0.2 wt Cu μg/g dry 371 1.9 9.4 224.8 37 266.4 190 135 5.0 17 41 69.2 18.5 113.9 5.0 27.9 5.0 0.88 3.6 165.3 1.4 2.0 Sample locations Mogpog Brgy. Malusak CB-08A-01 (0-35 cm) CB-08A-02 (35-70 cm) CB-08A-03 (70-115 cm) CB-08-01 (bottom) CB-08-02 (top) CB-05-01 (0-15 cm) CB-05-02 (15-30 cm) CB-04-01 (0-10 cm) CB-04-02 (10-15 cm) CB-03-01 (0-30 cm) CB-03-02 (30-59 cm) CB-03-03 (59-164 cm) CB-03-04 (164-188 cm) CB-12-01 (0-10 cm) CB-12-02 (147-177 cm) CB2-10-01 (0-20 cm) CB2-10-02 (107-120 cm) CB1-10-01 (0-100 cm) CB1-10-02 (100-147 cm) CB2-13-01 (0-50 cm) CB2-13-02 (50-115 cm) Table 7. Continuation Trace metal speciation in sediments 51

Conclusion and recommendation

This study was able to determine the spatial extent of metal contamination in Calancan Bay, Sta. Cruz, Marinduque as influenced by the presence of a tailings causeway from past mining activities. Total metal concentrations of Cu, Pb, Cr, and As revealed metal enrichment not only in areas closest to the tailings causeway but also in offshore areas of the bay. Speciation results indicated that copper present in sediment samples was found mostly in the organic fraction, while Pb was found to associate with the Fe/Mn oxides and hydroxides. Both Cu and Pb come from mainly anthropogenic inputs and that these two metals are in the mobile phases. As and Cr, on the other hand, were found in the lithogenic fraction of the sediment, suggesting natural origin.

The study used the traditional speciation scheme reported by Tessier et al. (1979) as this technique is robust and suits the objective of establishing the concentration levels of the different metals that could be bioavailable and/or may pose hazard to aquatic organisms. Recoveries from the Tessier et al.'s sequential extraction method were relatively quantitative for Cu and Pb. However, low recoveries were noted for As. The use of other speciation schemes, especially those that would give good recoveries for the speciation of Pb and Cr, is recommended.

Furthermore, comprehensive research will also be necessary to assess all factors affecting the mobilization, pathways, bioavailability and toxicity of heavy metals not only in Calancan Bay, Marinduque but also in other systems affected by mining operations. In particular, the conduct of aquatic toxicity testing is recommended to complement the speciation strategies employed in this study so as to quantify the toxicity of these different forms of metals to aquatic organisms. Recognizing the importance of determining the distribution and phase associations of trace elements in sediments via the selective sequential extraction scheme will not only avert possible hazardous and greater impacts but will also contribute in attaining sustainable development in the mining industry through responsible monitoring of mine wastes and tailings in the immediate environment.

Acknowledgment

The researchers are grateful for the financial and technical support provided by the following: the Lands Geological Survey Division of the Mines and Geosciences Bureau, Marine Geological Survey Division for assistance during sampling of core samples in Calancan Bay, and the Metallurgical Technology Division. The authors also acknowledge the assistance of Marnette Puthenpurekal, Jose Manipon, Enrique Maglanque, Robert Avellana, and Leopher Dagdag for the sampling, preparation of maps, etc. 52 D. Apodaca et al.

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Growth performance of three eucalyptus (Eucalyptus deglupta x E. pellita) hybrids on half-sib progeny trial in Northern Mindanao, Philippines

For. Albert A. Piñon Wilfredo M. Carandang, Ph.D. Science Research Specialist Assistant to the Chancellor and Ecosystems Research and Development Bureau Forestry Professor Department of Environment and Natural Resources University of the Philippines College, Laguna, Philippines Los Baños, Laguna [email protected]

Dr. Cesar C. Nuevo Forestry Consultant Cagayan de Oro City

Half-sib progeny trial as one essential aspect of Forest Tree Improvement to produce quality planting materials is limited in the Philippines. Using three F1 hybrid parents and 74 surviving F2 half-sib progenies of Eucalyptus deglupta x E. pellita hybrids, the economically important traits such as percent survival, total height, diameter at breast height and volume were evaluated. Comparison of the means using T-test analysis detected non- significant differences between border and inside trees. Outcome ofthe analysis of variance revealed that the three F2 half-sib progenies perform poorly due to the occurrence of inbreeding depression (ID), as reflected in a non-significant variation within and among the families in all phenotypic traits used, except in Total Height and Volume. Correlations indicated that future selection using either of the traits studied, particularly those with the highest value except for PS, can result in better volume harvest. Ranking

analysis proved the superiority of EDP1F2. It was followed by EDP3F2 then

EDP2F2. Results suggested that while ID negates the phenotypic superiority of Eucalyptus hybrids, application of appropriate silvicultural practices can correct its adverse effect.

Keywords: tree improvement, inbreeding, Eucalyptus, slope gradient, hybrid 56 Piñon et al. a slight increase in the forest cover of the philippines has been reported, yet its forests are still unstable and deforestation remains rampant (FAO 2010). To address this situation, identified solutions include protecting the remaining native forest and/or increasing re-vegetation through tree plantations. Several factors are contributing to the success or failure of any reforestation, but one key factor to successful tree plantations is the use of quality planting materials right from its establishment (Ng 1996). Correct planting materials at the very beginning coupled with appropriate silvicultural practices will ensure better quality plantation and profitable investment (Zobel and Talbert 1984; Nuevo 2001).

Quality Planting Materials (QPMs) are produced with parallel implementation of a strong Forest Tree Improvement Program (Zobel and Talbert 1984). Backed with this principle, the country had initiated some tree improvement projects as early as 1960s, together with the introduction of exotics like the Eucalyptus (Lizardo 1960). However, since tree improvement activities initiated were fragmented with the absence of serious implementation at the national scale, the Philippines does not have seed orchards, neither does it have efficiently managed Seed Production Areas (SPAs) nor a conscious effort to collect seeds from elite trees after 60 years (Carandang and Carandang 2004; ERDB 2012).

Fortunately, in support to the National Greening Program (NGP), the Philippine government is initially implementing a nationwide Research and Development Program on Forest Tree Improvement (RDP-FTI) through the Ecosystems Research and Development Bureau of the Department of Environment and Natural Resources (DENR-ERDB). This program aims to establish a phenotypically-improved seed source which includes SPAs, progeny test, provenance trial, seed orchard, clonal seed orchard, and individual plus trees (IPTs). Baseline information, therefore, particularly with regards to the establishment and assessment of a half-sib progeny test plantation is necessary to serve as guide for the implementation of RDP-FTI to produce the QPMs in support of the NGP until 2016 and beyond.

Along this line, a study was conducted to evaluate the growth performance of three half-sib progenies of 33-month-old Eucalyptus deglupta x E. pellita hybrids, which was established in Claveria, Misamis Oriental. Economically important traits assessed include percent survival (PS), diameter at breast height (DBH), total height (TH), and volume (VOL). Results were used to identify the best performing family of Eucalyptus hybrids used, and to determine the most appropriate silvicultural practices to attain optimum growth. Growth performance of three eucalyptus hybrids 57

Review of literature

Species description and phenology

Eucalyptus, together with the species of Acacia and Casuarina, is among the most important species for worldwide plantation due to inherent usefulness in various forms of wood utilization (Davidson 1995). It requires less intensive silvicultural interventions than other hardwoods, with established protocols for successful domestication, breeding, hybridization, and mass clonal propagation (Davidson 1995; Eldridge et al. 1993). Reproductive attributes of Eucalyptus are affected by numerous factors, including sexual system, mating system, flower bud initiation and development, fruiting phenology, pollinators and pollen dispersion, precocity, and fecundity. Eucalyptus exhibits mixed but predominantly out-crossing with partial mating system. Majority of the species are either wind-pollinated or insect-pollinated. Germinants per kilogram of seeds can range from 100,000-700,000 depending on the species used and germination management applied (Griffin 1989).

Despite being recognized as among the most important Eucalyptus hybrids in the Asia Pacific Regional Expert Consultation on Eucalyptus (Davidson 1995), a very limited number of publications has been reported to describe the botanical, ecological, geographical, and even economic importance of Eucalyptus deglupta x E. pellita. Preliminary result of the seedling performance of E. deglupta x E. pellita hybrids both in the nursery and field condition has been reported elsewhere (Siarot 1986 and 1991). Promising growth of this hybrid has already been proven, particularly by Bukidnon Forest Inc. (BFI) with collaboration to New Zealand government based from their two-year extensive species selection and seed origin suitability on various Eucalyptus (Razal et al. 2004). Le (2009) confirmed its suitability in Mindanao and mentioned the existence of this hybrid in the region.

Progeny trial

The usual basis of selection is done through visual observation (mass selection), assuming that phenotypically superior trees are also genetically superior. Normally, genetic testing is conducted using provenance trial. After the best provenance is identified, the best population of trees are evaluated and selected through progeny trial. Progeny or provenance trials are considered the beginning of most tree improvement programs. Progeny trial is a direct estimate of the parents’ genetic value, designed to describe the genetic variation within and between populations of known provenance trees (Zobel and Talbert 1984). When variation is found mostly genetic, small additional gain can be achieved, but if environmental, ample gains can be obtained from it (Van Buijtenen et al. 1976). 58 Piñon et al.

Progenies produced may be termed half-sib or full-sib. It is half-sib, when only a single parent is known (usually the female parent); otherwise, it is full-sib (Wright 1976). Essentially, it is designed to determine the degree of genetic strength, focusing mainly (but not purely) on the effect of genes. To some degree, permanency is infused to genes. But since genetic expression is bounded by the environment, progeny trial must be undertaken continuously as climate is infinitely changing (Wright 1976; Zobel and Talbert 1984; Puri 1998). Limited studies on progeny trial have been conducted in the Philippines. An example includes the study conducted by Haque (1995) on half-sib progeny test of Yemane (Gmelina arborea Roxb.) in Mindanao.

Hybrid and hybrid vigor

Hybridization does not intend to create new species but utilize the present extremities of characters between potential parents (Wright 1962, 1976; Zobel and Talbert 1984). Hybrid vigor or heterosis is an inherent trait that is always correlated to the environment where the hybrid is growing (Moore et al. 2003). Generally, hybrids can maintain, destroy, or create the species diversity through reproductive isolation, reinforcement and formation of hybrid swarm, introgression, and increased adaptation, respectively (Judd et al. 2002). In many cases, hybrid shows some superiority in areas where either of the parents is unable to express their full potential. For example, hybrid of Eucalyptus grandis x E. teriticornis in Congo have wood yield at 40 m3 ha-1 yr-1, where E. grandis parent is not adapted (Martin 1989).

Methodology

Experimental site

The open-pollinated half-sib progeny test (about 3000 m2) used in this study with an elevation ranging from 300-400 masl is a portion of 1.2 hectare plantation established on August 2008 by the Claveria Tree Nursery, Inc. (CTNI) in Barangay Gumaod, Claveria, Misamis Oriental (lat 8.66°N, long 124.8°E) (Figure 1, Tables 1 and 2). This progeny trial is composed of one family of Eucalyptus deglupta and three different families of E. deglupta x E. pellita hybrids. Test site is surrounded by Teak (Tectona grandis) and the undergrowth vegetation is dominated by Coronitas (Lantana camara), Hagonoi (Wedelia biflora), and Wild Sunflower Tithonia( diversifolia). Previous agricultural crops that were intermittently cultivated in the area were Corn (Zea mays) and Cassava (Manihot esculenta). Based on PAGASA’s climatic data generated from Lumbia weather station (2008-2011), the minimum, maximum, and mean annual rainfall (MAR) were 1800, 2352, and 2112 mm, respectively. While the minimum, maximum, and mean annual temperatures (MAT) were 26.4°C, 27.5°C, and 27°C, respectively. Growth performance of three eucalyptus hybrids 59 and three f Eucalyptus deglupta Line number hybrids at CTNI, Claveria, Misamis Oriental (red dot); and F2 F2 F2 F2 E. deglupta x pellita EDP1 EDP2 EDP3 ED hybrid plantation in Kitcharao, Agusan del Norte where parents are planted H ills Number of Planting layout of half-sib progeny test one family o families of Eucalyptus (yellow dot) in the Philippine map (2012). Line number Figure 1. 60 Piñon et al.

Table 1. Site description of half-sib progeny test of E. deglupta x E. pellita hybrids in Barangay Gumaod, Claveria, Misamis Oriental based on 2012 data collection. Description Site and climatic data Location Claveria Tree Nursery, Inc. (CTNI), Claveria, Misamis Oriental Area 3000 m2 Latitude 8.66° N Longitude 124.8° E Elevation 300 - 400 masl Mean Annual Rainfall 2112 mm Mean Annual Temperature 27°C Mean Annual Relative Humidity 83% Soil Silty clay Sand 5.78% Silt 34.33% Clay 59.89% pH 4.61% Organic Matter (OM) 3.68% Nitrogen (N) 0.15% Phosporus (P) 0.611 ppm Potassium (K) 0.473 me/100 g soil Planting date August 2008 Number of families 3 Fertilizer applied Mixture of inorganic and organic compost (once) Plant spacing (Quincunx) 4.2 m within row x 4.3 m between rows Total number of seedlings planted 172 (no replanting for mortality) Slope gradient 0 - 9 degrees Growth performance of three eucalyptus hybrids 61

Table 2. Site description of three half-sib mother trees of E. deglupta x E. pellita hybrids from Kitcharao, Agusan del Norte based on 2011 data collection. Description Generated data Note Year planted 1998 Mean Annual Rainfall 2170.6 mm Mean Annual Temperature 28.1 °C Mean Annual Relative Humidity 84% Aspect NE Light exposure Full Topography Rolling Surrounding trees Eucalyptus deglupta; Paraserianthes falcataria; Artocarpus blancoi Distance from each other (m)

EDP1F1 and EDP2F1 23.9

EDP1F1 and EDP3F1 28.2

EDP2F1 and EDP3F1 5.9 Relative slope position

EDP1F1 High

EDP2F1 Low

EDP3F1 Low DBH (cm)

EDP3F1 55.7 Uncut Stump diameter (cm)

EDP1F1 50 Cut in 2010

EDP2F1 55 Cut in 2010 Total height (m)

EDP1F1 3 Coppice

EDP2F1 11.74 Coppice

EDP3F1 26.9

Merchantable height of EDP3F1 15.2 m

Crown area of EDP3F1 (m) East West (EW) 18.2 North South (NS) 13.8 62 Piñon et al.

On the other hand, recorded relative humidity varied from 82-85%, with highest mean annual relative humidity (MRH) of 85% in 2011.

Soil physical and chemical properties

A composite of eight soil samples per slope gradient from 0-30 cm depth was collected from the experimental site. Soil samples were analyzed using the routine soil analysis at the Analytical Service Laboratory, Soils and Agro-Ecosystems Division of the College of Agriculture, University of the Philippines Los Baños. Results revealed that the soil in the trial is silty clay with average textural classes of 5.78%, 34.33%, and 59.89% for sand, silt and clay, respectively (Table 3).

Table 3. Average soil physical and chemical properties of F2 half-sib progeny test of E. deglupta x E. pellita hybrids at CTNI, Claveria, Misamis Oriental. K Slope OM N P (me/ Family Sand Silt Clay pH Gradient (%) (%) (ppm) 100 g soil)

EDP1 SL 1 40 59 4.6 3.62 0.15 0.5 0.38

SM 11 32 57 4.7 3.67 0.14 0.4 0.41

SH 8 38 54 4.7 3.37 0.15 0.3 0.57

EDP2 SL 3 35 62 4.5 3.83 0.16 0.7 0.39

SM 8 32 60 4.5 3.89 0.17 0.7 0.41

SH 8 32 60 4.5 3.86 0.15 1.4 0.43

EDP3 SL 6 32 62 4.5 4.16 0.17 1 0.48

SM 2 33 65 4.6 3.24 0.14 0.1 0.60

SH 5 35 60 4.7 3.52 0.14 0.4 0.59

Slope determination

Slope gradient was determined using an abney-hand level and calibrated height-pole. Measurement was done once every other line in every fifth tree (about 21 m) from the baseline up to the last tree of that line (north-south) between tree line numbers 25 to 35. Collected data were plotted and fitted to the plantation map, followed by an interpolation to come up with the relative distances among three slope gradients. Accordingly, slopes were stratified into <3, 3.1 to 6, and 6.1 to 9 degrees, which were noted as low, medium, and high slope gradient, respectively (Figure 2). Growth performance of three eucalyptus hybrids 63

<30

3.1 -60

>6.10

Mortality

Surviving Progeny Number of Surviving Progenies Line number

Figure 2. Surviving F2 progenies in various slopes of half-sib progeny test of E. deglupta x E. pellita hybrids at CTNI, Claveria, Misamis Oriental based on 2012 data collection.

Plant material

Seeds from three half-sib mother trees of E. deglupta x E. pellita hybrids were collected from plantation under the Usufruct Program by the Department of Environment and Natural Resources (DENR-13), which was established in Kitcharao, Agusan del Norte in January 2008. The planting materials used were supplied by the Provident Tree Farms, Inc. (PTFI) (personal oral communication with C Nuevo; unreferenced). Nuevo further mentioned that this Eucalyptus is a product of natural hybridization between E. deglupta (Surigao, Mindanao) and E. pellita (Australia), which was originally acquired by PTFI. 64 Piñon et al.

On February 2008, the seeds were germinated. Seedlings produced were raised and maintained at CTNI until they have reached the plantable size. In August 2008, after culling or removal of phenotypically inferior seedlings, the total 172 seedlings produced were planted: 51 progenies each for EDP1F2 and EDP3F2, while

70 for EDP2F2. No replanting was done to replace the mortality.

Experimental design

Using a Randomized Complete Block Design (RCBD) with unequal number of experimental units, the analysis of variance (ANOVA) and correlations for all surviving progenies were calculated for traits such as percent survival (PS), diameter at breast height (DBH), total height (TH), and volume (VOL). Calculation was made using SAS GLM Procedure Program version 9.1 of 2012. The following mathematical model was used:

Yijk - μ + Bi + Fj + BFijk

where:

Yijk - Phenotypic value of the kth tree of the jth family in the ith block μ - Population mean

Bi - effect of the ith block

Fj - effect of the jth family

BFijk - error effect

Tree parameters measured

Phenotypic parameters were measured last May 2011. DBH values were computed by dividing the circumference (C) reading to the value of π. C was collected using the meter tape at 1.3 m above-ground. Meanwhile, TH was obtained by using a calibrated height-pole, while DBH and VOL were calculated using the raw data for C and TH. Derived DBH and TH measurements were then used in calculating each volume. Finally, PS for each slope and family was computed by counting the number of surviving progenies over mortality.

Data analysis

To detect any significant differences between border and inside trees, comparison of the means using T-test was done with the assumption that there was no significant difference in growth traits of trees between the edges and inside location (Figure 3). Analysis of variance was then applied for all the traits used. The significant differences between treatment means were tested using the Least Significant Difference (LSD) Test. Growth performance of three eucalyptus hybrids 65

Border Trees

Inside Trees

Mortality

Surviving Progeny Number of Border & Inside Progenies Line number

Figure 3. Randomly selected surviving F2 half-sib progenies of E. deglupta x E. pellita hybrids between border and inside locations at the experimental site, CTNI, Claveria, Misamis Oriental based on 2011 data collection. Best family

Simple ranking analysis using a point system that ranged from 1 (lowest) to 3 (highest) per variable was used to identify the best performing hybrid family based from the mean phenotypic characters. In this study, family with the highest accumulated points was considered superior over the others.

Results and discussion

Border vs. Inside Trees

Results of the T-Test analysis among phenotypic traits (DBH, TH, and VOL) revealed that regardless of slope position, surviving progenies of three E. deglupta x 66 Piñon et al.

E. pellita hybrids were insignificantly different from each other (Table 4). This would indicate that the influence of non-genetic factors between edges and inside location was not significant to provide some detectable phenotypic variation among progenies.

Table 4. Result of T-test analysis between border and inside trees Traits Mean Std. Error T-Value Pr > |t| DBH (-) 0.552 0.608 (-) 0.91ns 0.379 TH (-) 0.973 0.626 (-) 1.55 ns 0.143 VOL (-) 0.007 0.007 (-) 1.17 ns 0.261 ns - not significant Percent survival

Computed overall mean PS was very low at 44.88% with no significant difference in variation due to family and slope gradient, both within and among F2 progenies (Tables 5, 6, and Figure 4). Non-significant variation due to family indicated closer inherent characteristics of the F2 hybrids. Eucalyptus’ mating system such as ‘selfing’ (cross-pollination among flowers of the same tree) and ‘kin-mating’ (cross-pollination among relatives) normally resulted in inbreeding or narrowing of the genetic base (Griffin 1989). Thus, inbreeding depression (ID) reduced the performance of three hybrid progenies. Martin (1989) explained that forest trees are often allogamic and that heterozygotes hide a number of half-lethal genes. Based on Hardner and Potts (1995), ID tends to increase with age, and culling ‘selfs’ in the nursery is not an option as inbreeding effects may become significant as early as eight months after planting in the field. However, although Griffin (1989) claimed that ‘selfing’ and ‘kin-mating’ are some possible reasons of inbreeding amongEucalyptus , the main causes of ID that occurred among F1 hybrid parents cannot be specified, since controlled pollination to clearly discriminate the mode of inbreeding has not been considered in this study.

Perhaps, the work of Siarot (1991) cements the authors' previous claim that the hybrid parents used in this study undergone inbreeding, hence F2 shows a depressed survival. Percent survival obtained in the study was very low compared to what has been reported by Siarot (1991), who achieved an overall average of 71.3% from various F1 Eucalyptus hybrids after 36 months. Reported percent survival of the other Eucalyptus like E. camaldulensis (75%) and E. tereticornis (76%) in Pasuquin, Ilocos Norte, 60 months after planting (Malab 1996) were also relatively higher than this study. Similarly, results of this study is far behind Portugal’s E. globulus that recorded a percent survival of up to 90%, 36 months after planting (Borralho et al. 1992). In contrast, the result of the study is higher than the 32% survival obtained from Magat Reforestation Project using E. camaldulensis in Nueva Vizcaya, Philippines (Maun 1978). Growth performance of three eucalyptus hybrids 67

Table 5. Mean growth performance within family of F2 half-sib progenies of E. deglupta x E. pellita hybrids. Traits Family Percent Diameter at Breast Total Volume (m3) Survival (%) Height (cm) Height (m)

EDP1F2 51.46 6.70 5.47 0.032

EDP2F2 47.13 6.01 4.73 0.0171

EDP3F2 36.06 6.53 6.41 0.0252 Mean 44.88 6.42 5.54 0.025 Pr > F 0.2151 0.6159 0.3888 0.6574 CV 20.16 23.62 19.33 57.81

Table 6. Mean growth performance among families of F2 half-sib progenies of E. deglupta x E. pellita hybrids.

Traits Family Percent Diameter at Breast Total Volume (m3) Survival (%) Height (cm) Height (m)

EDP1F2 51.46 6.70 5.47 0.032

EDP2F2 47.13 6.01 4.73 0.0171

EDP3F2 36.06 6.53 6.41 0.0252 Mean 44.88 6.42 5.54 0.025 Pr > F 0.2151 0.6159 0.3888 0.6574 CV 20.16 23.62 19.33 57.81

Diameter at Breast Height (DBH)

Both variations due to family and slope gradient within and among families were statistically insignificant (Tables 5, 6, and Figure 4). This is probably the effect of ID, such that the optimal growth to command detectable differences on DBH was not achieved. Hardner and Potts (1995) reported that ID severely affects field growth of E. globulus ssp. relative to outcrossing, and that the effects on diameter growth appeared later than height. Nonetheless, the diameter growth recorded was quite higher than those reported for trial planting of E. deglupta (10.2 cm) in Bukidnon 48 months after planting (Lizardo 1960), and slightly lower than E. deglupta intercropped with maize (15.6 cm after 54 months) in northern Mindanao (Bertomeu 2003). In contrast, computed mean DBH (6.42 cm after 33 months) in this study was relatively higher than the best Eucalyptus provenance (7.9 cm after 60 months) in New Zealand (Millner 2006). 68 Piñon et al.

a ab b

Figure 4. Mean PS, DBH, TH, and VOL within (family) and among (slope) half-sib progenies of E. deglupta x E. pellita hybrids. Bars (Vol) with the same letter/s are not significantly different.

Total Height (TH)

Variation due to family was statistically insignificant, while among families, mean TH varied significantly P( = 0.005) (Tables 5, 6, and Figure 4). This suggests that favorable microclimate condition at varying slope positions overrides the effect of ID after 33 months. Growth performance of three eucalyptus hybrids 69

The above-mentioned suspected ID brought by relative genetic uniformity and possible water stress condition in the study site, explicitly showed why like the mean values derived from other phenotypic traits, TH did not vary significantly among hybrid families due to family. In contrast, significant variation across slope gradients indicate that although ID occurs earlier in height than diameter growth (Hardner and Potts1995), favorable microclimate condition tends to disclose the effect of ID causing significant variation on height growth. This is attributed to non- genetic factors that also control the expression of continuous characters (i.e. TH) (Moore et al. 2003). Hence, even the growth performance of Eucalyptus clones with the same genetic characteristics varies depending on the available resources, such as water and nutrients (Boyden et al. 2008). Since internal physiological processes, which are responsible for tree growth, vary depending on trees’ genetic potential and availability of resources required, the potential for optimal growth will never be achieved fully (Kozlowski and Pallardy 1997, Pallardy 2008). Calculated overall mean height growth of E. deglupta x E. pellita hybrids in the study site was almost equal to E. deglupta (7.89 m after 48 months) reported by Lizardo (1960), but relatively taller than measured in E. deglupta (2.17 after 16 months) at Costa Rica (Cornelius et al. 1995).

Volume

Like other traits, family differences were also found insignificant, while variation due to varying slope was found significant P ( =0.027) (Tables 5, 6 and Figure 4). Differences demonstrated by family were insignificant. This result supports the study of Marco and White (2002), where variation due to family in volume growth of E. grandis and E. dunnii was also found insignificant after 36 and 72 months, respectively. In contrast, there was a significant difference detected from mean VOL due to slope gradient, implying that significant variation detected in height growth was strong enough to also influence the volume growth. Volume as a function of DBH and height depends mainly on these traits (Nuevo 2001, Wright 1962, Zobel and Talbert 1984), and therefore, any significant effect that can be observed from either of these traits will have a repercussion in volume performance. Since trees situated at SM was found significantly different from those surviving at SL, but not in

SH, best performers at SL of EDP1F2 imply better future yield than trees of other slopes.

Correlation analysis

Weak and insignificant correlation was observed between PS and other phenotypic traits (Table 7), which suggests that phenotypic selection based from survival ability is not a good parameter to achieve better future volume yield. In contrast, significant and very strong positive correlation was obtained between DBH 70 Piñon et al.

Table 7. Correlations among phenotypic and genetic characters of three F2 half-sib progenies of Eucalyptus deglupta x E. pellita hybrids at CTNI, Misamis Oriental. SUR DBH HT VOL H' L P B SUR 1.000 -0.183 -0.268 0.015 0.645 0.705 0.705 0.689 0.637 0.486 0.970 0.061 0.034 0.034 0.040 DBH 1.000 0.700 0.955 -0.225 -0.265 -0.265 -0.043 0.036 <0.0001 0.561 0.492 0.491 0.250 HT 0.734 -0.464 -0.633 -0.633 -0.573 0.024 0.208 0.067 0.067 0.107 VOL 1.000 -0.186 -0.221 -0.221 -0.338 0.632 0.568 0.568 0.373 Note: Bold red figure is highly significant, while bold black values are significant, and italized figure is the probability value. and VOL (r = 0.955; P< 0.0001). Significant and strong positive association was also detected between DBH and TH (r = 0.669; P = 0.036). Such results indicate that better volume harvest can be guaranteed by using either of these traits as basis for future selection. Almost the same correlation result between DBH and TH (r = 0.70) of E. urophylla has been reported in northern Vietnam (Kien et al 2009). Strong and positive correlation between DBH, TH, and VOL has long been recognized by many tree improvers (Wright 1962, Zobel and Talbert 1984). They suggested that fertilizer application, which is intended to increase the stem diameter, can actually also improve both the volume and total height growth. This is supported by Ginwal et al. (2004), where they found that phenotypic correlations among height, clear stem height, and collar diameter of E. tereticornis were highly significant. This led to the conclusion that applying tree improvement can improve all these traits simultaneously.

Best family

Ranking analysis demonstrated the superiority of EDP1F2. It was followed by

EDP3F2 then EDP2F2 (Table 8).

Conclusion

Results of the analysis on half-sib progeny test revealed that non-significant variation due to family on all phenotypic traits implied that Eucalyptus deglupta x E. pellita hybrids used have undergone ‘selfing’ or any other form of inbreeding, hence the demonstration of Inbreeding Depression (ID) in their half-sib progenies. Growth performance of three eucalyptus hybrids 71

Table 8. Ranking of growth performance of three Eucalyptus deglupta x E. pellita hybrids at various slope gradients. Family Slope gradient Characters EDP1F2 EDP2F2 EDP3F2 SL SM SH Phenotypic Percent Survival 3 2 1 2 3 1 Diameter at Breast Height 3 1 2 3 1 2 Total Height 2 1 3 3 1 2 Volume 3 1 2 3 1 2 Total 11 5 8 11 6 7

Apparently, the hybrid superiority of the parents was not fully demonstrated due to the effect of ID. But since significant variation was observed among families in terms of TH and VOL, it appears that favorable microclimate condition as contributed by varying slope positions counterbalanced the negative effect of ID. This suggests that manifestations of ID are correctible through the application of appropriate silvicultural practices (i.e., fertilization). The result of phenotypic correlations generally revealed that PS, as one important trait, was not directly affected by the other traits. This implies that using the hybrid family with the highest percent survival as the basis for selection for future plantation does not guarantee a better volume yield. Finally, positive with strong to very strong and statistically significant associations between DBH, TH, and VOL emphasized that better future volume harvest can be obtained from using the hybrid family (Eucalyptus deglupta x E. pellita) with the highest growth performance on any of these traits.

Recommendations

Based on the result of the study, the following actions are recommended:

1. Apply appropriate silvicultural practices (i.e., fertilization) to improve the growth performance of the E. deglupta x E. pellita hybrids half-sib progeny test. 2. Using DBH as the basis for selection, collect the seeds of the best half-sib progenies among the three hybrids for future genetic improvements. 3. Collect the seeds from other proven phenotypically-improved Eucalyptus (i.e., E. deglupta) and infuse/combine these with the seeds from the best performing hybrid progenies used in the study for the next cycle of open- pollinated half-sib progeny test. 4. If deemed useful for future plantation, cuttings from the identified 72 Piñon et al.

phenotypically and genetically superior hybrid progenies can be collected for macrosomatic cloning. 5. Replicate the same study with other fast-growing exotic and native forest tree species (i. e., E. deglupta, Paraserianthes falcataria, Vitex parviflora, etc.)that is relevant to the implementation of the National Greening Program.

Acknowledgment

The primary author is indebted to the following: Department of Science and Technology-Science Education Institute (DOST-SEI) for Masteral Program Scholarship and Thesis Grant for enabling the primary author to pursue graduate studies; Chairman of the Committee of his advisers, Dr. Wilfredo Carandang for his unwavering guidance throughout this work; the ERDB Assistant Director Leuvina M. Tandug for her helpful advice in statistics; CTNI, RI Chemical family, and the rest of field and nursery staff for allowing him to use their Eucalyptus progeny trial; Dr. Cesar Nuevo, his greatest mentor, role model, friend, and second father; Above all, to Jehovah God for the life and His absolute love.

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Assessment of tree species diversity and structure of selected government reforestation projects

For. Manolito U. Sy Alexander John D. Borja Supervising Science Research Specialist Science Research Analyst Forest Ecosystems Research Division Ecosystems Research and Development Bureau For. Rosalinda S. Reaviles 4031 College, Laguna Senior Science Research Specialist [email protected] Marcelina V. Pacho For. Jose Alan A. Castillo Science Research Specialist II Science Research Specialist 1 For. Roy Joven R. Amatus Science Research Analyst

This study investigated and analyzed the composition and structure of three government reforestation projects: Paraiso Reforestation Project (PRP) in Piddig, Ilocos Norte; Nassiping Reforestation Project (NRP) in Gattaran, Cagayan; and Marinduque Reforestation Project (MRP) in Boac and Torrijos, Marinduque. Purposive sampling was applied using 50 m X 50 m sampling plots established within the plantations considered mature or old.

A total of 165 species belonging to 120 genera and 49 families were recorded in the three reforestation projects. Swietenia macrophylla King was found either dominant or co-dominant in the three reforestation sites but pose a potential threat for bio-invasion.

The diversity level in PRP, NRP, and MRP based on Shannon-Wiener Index were computed at 2.77, 3.01, and 3.13, respectively. By comparison, the values of diversity index ranges from Moderate to High based on modified Fernando Biodiversity Scale (1998). The computed values for Simpson’s Index of Dominance for PRP, NRP, and MRP were 0.84, 0.90, and 0.88, respectively, indicating the dominance of only few tree species in the entire community, notably the tree species such as S. macrophylla, Reutealis trisperma (Blanco)

Keywords: species diversity, structure, reforestation project, sampling 76 Sy et al.

Airy Shaw, Tectona grandis L.f., Adenanthera intermedia Merr., Gmelina arborea Roxb., Vitex parviflora Juss., and Pterocarpus indicus Willd. forma echinatus.

Out of the 165 species, there were 101 indigenous, 38 endemic, and 27 introduced species. Among the species listed as endemic or indigenous, a total of 21 species recorded in the three study sites were listed in the National List of Threatened Philippine Plants of which four species are Critically Endangered including R. trisperma and Toona calantas Merr. & Rolfe, six species are Endangered, seven species are Vulnerable, and four species are listed as Other Threatened Species. Most of the species listed are timber-producing trees and economically important for furniture and high quality lumber.

Rehabilitation efforts through reforestation activities in the Philippines started when the School of Forestry (now College of Forestry and Natural Resources) was established in 1910. Since then, several reforestation projects were established involving the government, private companies/individuals, NGOs, people’s organizations, and local communities to restore denuded areas. These include regular reforestation projects covering an aggregate area of about 1.4 million hectares of which 0.36 million hectare or roughly 25% has been reported to have been planted with one or more kinds of plant species. These government reforestation projects, totaling about 250, were established as early as 1916 and continued until 2000.

However, government-initiated reforestation activities were not given much emphasis and serious consideration due to insufficient funds for their management. Thus, the reforested areas were left at the care of nature unprotected and unmanaged. If left intact, forest plantations or reforestation areas would eventually develop into a multi-species stand and could become a haven of diverse plant and animal species.

Three of these reforestation stands were considered in this study. Since their establishment, none has been done and reported to assess their current status. Specifically, this paper would: 1) Assess the floristic composition of three government reforestation projects to provide updated information on the nature and status of their floral species as input information for preparing plans and programs for their management; 2) Present the quantitative structure of the tree species found in each of the three reforestation sites; and 3) Identify useful and threatened species for conservation management and possible source or gene banks for rare, vulnerable, and endangered indigenous tree species. Tree species diversity and structure of selected government reforestation projects 77

The information generated by the study could be used to inform decision and policy making as regards the management of the country’s old and current reforestation stands as well as input to making plans and programs.

Review of literature

Vegetation is an important part of the ecosystem. It is the most obvious physical representative of the ecosystem. It acts as habitat of organisms within which other organisms live, grow, reproduce, and die (Kent and Coker 1992). The description of vegetation normally involves an account of its floristic composition or in combination with its structure (Mueller-Dombois 1974 as cited in Vermudo 1995).

Vegetation analysis determines the most important species in the community. The importance could be in terms of density, cover, and frequency or their combination. The degree of importance of a species may reflect its influence to the biotic community. This is based from the “a priori” assumption that the less represented species have poor influences in terms of the utilization of energy flowing to the community (Vermudo 1995).

The major aim of vegetation studies is concerned with the identification of plant communities and its constituent flora and the link or relations that may exist among them, to provide the basic description and characterization of the existing vegetation. Such aim could allow the formulation of hypothesis about the major direction and variations of vegetation development, and how such variations are being influenced by environmental factors (Hunley and Birks (1979) as cited by Vermudo (1995).

Likewise, information on vegetation may be required to address certain ecological inquiry (e.g., biological conservation and management), on meeting requirements for environmental impact assessments, on monitoring of management practices, and on providing the bases for the prediction of possible scenarios (Kent and Coker 1992). Knowing the present status of the vegetation is the first step in any resource-based management planning (Vermudo 1995).

Methodology

From the list of regular government reforestation projects requested from the Forest Management Bureau (FMB), three reforestation sites were identified for study. These were the Paraiso Reforestation Project (PRP) in Piddig, Ilocos Norte; the 78 Sy et al.

Nassiping Reforestation Project (NRP) in Gattaran, Cagayan; and the Marinduque Reforestation Project (MRP) in Boac and Torrijos, Marinduque. Figure 1 shows the relative locations of these project areas. The study was conducted for a year, from April 2010 to March 2011.

Site description

The PRP is under the management of the DENR, specifically Provincial Environment and Natural Resources Office (PENRO) of Ilocos Norte and Community Environment and Natural Resources Office (CENRO) of Laoag City. According to information obtained from the said CENRO, it was established on 07 May 1930 by the late Forestry Supervisor Espiritu Paraiso, a native of Piddig, Ilocos Norte. It was originally named Tangaoan Reforestation Project because the project’s main nursery and office was situated within the Barangay of Tangaoan, Piddig, Ilocos

Figure 1. Map of the Philippines showing the relative locations of the Project Sites Figure 1. Map of the Philippines showing the relative locations of the project sites. Tree species diversity and structure of selected government reforestation projects 79

Norte but it was changed to the present name through a Resolution passed by the Piddig Municipal Council when Mr. Paraiso died in 1937. According to the project profile provided by the CENRO, the PRP covers an area of 7,001 ha with 1,721 ha of established plantations spread in four other barangays in the same municipality and in one barangay in the adjacent Municipality of Carasi. The site is under Climatic Type I based on Modified Coronas Classification, where there are two pronounced seasons: dry from November to April and wet during the rest of the year.

On the other hand, the NRP covers an area of 2,011 ha consisting of natural secondary growth forest, agricultural areas, and plantation forests traversing from Barangay Nassiping, Gattaran to the adjacent Municipality of Alcala. According to the records obtained from FMB (2009), the whole project area was planted from 1939 (the year the project was established) up to 1991. NRP is jointly managed by the Department of Environment and Natural Resources (DENR) and the Provincial Government of Cagayan (LGU) by virtue of a Memorandum of Agreement (MOA) signed on 08 September 1998. The project site is under Type III Climate where there is no very pronounced maximum rain period, with dry season lasting only a maximum of three months.

MRP was established in 1937 with a total area of 4,885 ha, 3,991 ha of which had been planted with narra, mahogany, yemane, ipil-ipil, acacia, teak, and talisai from establishment to 1990 (FMB 2009). MRP had its central nursery and project office in Barangay Dampulan, Torrijos, Marinduque where tree plantation was reportedly established like in five other areas within the same municipality and in the Municipality of Boac. Like the NRP, the site belongs also to Type III Climate.

Establishment of sampling plots

Purposive sampling was applied in gathering data from the reforestation project. Sampling plots measuring 50 m x 50 m were randomly established following the cardinal direction within the plantation of mature stands. Within each sampling plot, all trees with diameter at breast height (DBH) of 5 cm and larger were identified, tallied, and measured as to their merchantable height (MH, for trees 10 cm dbh and larger only) and total height (TH).

Data analysis

Scientific names are after Rojo (1999) and family names are from Fernando (1999). The data were analyzed to determine parameters such as density, frequency, basal cover, and importance value as discussed in Kent and Coker (1992). 80 Sy et al.

The corresponding basal area of each individual tree is computed using the following formula:

Basal area (BA, sq m) = 0.00007854*DBH2; DBH in cm

The density value is represented by the number of individuals of a species per unit area and dominance value is computed as the sum of basal area values per species. On the other hand, the frequency for each species is given by the formula:

Frequency = Number of occurrence among the n plots n plots

The relative values are given by the following formulas:

Relative density = Density of a species Density of all species * 100

Relative dominance = Dominance of a species Dominance of all species * 100

Relative frequency = Frequency of a species * 100 Frequency of all species

The importance value (IV) of a species is given by the formula:

IV = Relative density + Relative dominance + Relative frequency

The data were also transformed to determine the species diversity indices. Index of species diversity and dominance index were also evaluated using Shannon- Wiener Index (H´) and Simpson’s index (C), respectively, with the following formula (Kent and Coker, 1992):

H' = - pi * ln pi

where: H = Shannon Index of Species diversity

pi = proportion of abundance of ith species relative to all species (relative abundance)

ln = log basen Tree species diversity and structure of selected government reforestation projects 81

2 C = n i

where: C = dominance index or Concentration of Dominance

ni = relative abundance of each species

Using the Shannon Index, the measure of evenness (J) can also be computed using the equation below (Kent and Coker 1992):

J = H'/ln (S)

where: J = evenness index H’ = Shannon Index of Species diversity S = number of species recorded

ln = log basen

Evenness (J) is a measure of how even are the species in the quadrat. The higher the value of J, the more even the species are in their distribution within the quadrat (Kent and Coker 1992). An evenness that is equal to one means all species are equally represented in the community.

The computed H’ index was interpreted based on a scale modified from Fernando Biodiversity Scaling system (Fernando, 1998) as shown in Table 1.

Table 1. Modified Fernando Biodiversity Scale.

Relative values Shannon-Wiener index (H') Very high 3.5-4.0 High 3.0-3.49 Moderate 2.5-2.99 Low 2.0-2.49 Very low 1.99 and below

Scientific and Family names are after Rojo (1999) and Fernando (1999). The conservation status of each species was evaluated using the National List of Threatened Philippine Plants and Their Categories, and the List of Other Wildlife Species based on the following conservation status as stated in DAO 2007-01, to wit: 82 Sy et al.

a. Critically Endangered – refers to a species or subspecies facing extremely high risk of extinction in the wild in the immediate future. This shall include varieties, formae, or infraspecific categories.

b. Endangered – refers to a species or subspecies that is not critically endangered but whose survival in the wild is unlikely if the causal factors continue operating. This shall include varieties, formae, or other infraspecific categories.

c. vulnerable – refers to a species or subspecies that is neither critically endangered nor endangered but is under threat from adverse factors throughout its range and is likely to move to the endangered category in the future. This shall include varieties, formae, or other infraspecific categories.

d. other Threatened Species – refers to a species or subspecies that is not critically endangered but whose survival in the wild is unlikely if the causal factors continue operating. This shall include varieties, formae, or other infraspecific categories.

Results and discussion

Floristic composition

A. Paraiso Reforestation Project (PRP)

The data gathered recorded a total of 993 trees per hectare belonging to 74 species (including two species of palm), 57 genera and 26 families (Figures 2 and 3). Family-wise analysis of species revealed that Fabaceae was dominant with 15 species, followed by Moraceae (nine species), Anacardiaceae (six species), Euphorbiaceae, and Sapindaceae (five species). The other 21 families were represented by less than four species each.

Fabaceae was also observed to have the most number of genera identified (14 genera), followed by Anacardiaceae, Moraceae and Sapindaceae (four genera), Apocynaceae, Euphorbiaceae, and Lauraceae (three genera), and Arecaceae, Meliaceae, and Rubiaceae (two genera). Other 16 families were represented by one genus each. The complete listing of species found and identified in PRP is shown in Appendix Table 1. Tree species diversity and structure of selected government reforestation projects 83

Figures 2 and 3. The Paraiso Reforestation Project (established in 1969) in Barangay Tangaoan, Piddig, Ilocos Norte. Inside are the Tectona grandis plantation (left) and Reutealis trisperma plantations (right).

B. Nassiping Reforestation Project (NRP)

A total of 67 species belonging to 50 genera and 28 families were recorded. The complete listing of species is shown in Appendix Table 2. The three most diverse families collected across all plots were Fabaceae, Anacardiaceae, and Lamiaceae (Figures 4 and 5). Among families, Moraceae (14 species) was the most species diverse. Fabaceae was represented by six species, Anacardiaceae, Euphorbiaceae, and Lamiaceae by five species, Meliaceae and Sapotaceae by three species, and Rutaceae, Tiliaceae, Burseraceae, Clusiaceae, and Malvaceae by two species. The remaining 16 families were represented by only one species each.

In terms of genera, Fabaceae was most represented with six genera, followed by Anacardiaceae (five genera), Lamiaceae (four genera), Moraceae, Euphorbiaceae, Meliaceae, and Sapotaceae (three genera each), Rutaceae and Tiliaceae (two genera each). The rest of the families were represented by one genus. 84 Sy et al.

FiguresFigures 4 4and and 5. 5 .The The teak plantation at at NRP Nassiping where 71Reforestation trees/ha were Project noted whereto have 71 trees/hadefects were or damages noted tosuch have as stemdefects cavities, or damages butt rots such and asbroken stem cavities,stems. butt rots and broken stems.

C. Marinduque Reforestation Project (MRP)

A total of 89 species (including one species of palm), belonging to 69 genera and 35 families, were recorded from two sites: Barangay Dampulan, Torrijos and Barangay Tumagabok, Boac, Marinduque. The listing of species observed in MRP is shown in Appendix Table 3.

Families Fabaceae and Moraceae had the highest (13) number of species, followed by Euphorbiaceae (6), Dipterocarpaceae, Meliaceae, and Sterculiaceae (4), Annonaceae, Apocynaceae, Lamiacaea, Lauraceae, and Ulmaceae (3) and the remaining 27 families were represented by less than two species each.

On the generic level, family Fabaceae was the most dominant represented by 12 genera followed by Euphorbiaceae and Meliaceae (four genera), Moraceae, Sterculiaceae, Annonaceae, Apocynaceae, Lamiaceae, and Lauraceae (three genera), Dipterocarpaceae, Ulmaceae, Lythraceae, Rutaceae, and Sapotaceae (two genera) (Figure 6 and 7). The other remaining families were represented by a single genus.

By comparison, MRP recorded the most number of species, genera and families (Table 2). Species composition recorded in PRP and NRP were almost the same in number.

Enumeration of families, genera, and species in the three reforestation projects showed the presence of 46 families, 109 genera, and 165 species, including three species of palm, distributed in a wide range of elevation between 22-544 meters above sea-level (masl). Fabaceae was observed to be the most diverse and common in terms of genera and species in all sites. Tree species diversity and structure of selected government reforestation projects 85

Figure 6. Measuring the DBH of a tree at Marinduque Reforestation Project, Dampulan, Torrijos, Marinduque. 86 Sy et al.

Figure 7. Forked and butt rotten narra at Marinduque Reforestation Project, Dampulan, Torrijos, Marinduque.

Table 2. Species composition and density in the three reforestation projects.

Reforestation project Species Genera Families recorded recorded recorded Marinduque Reforestation Project 89 69 35 Paraiso Reforestation Project 74 57 26 Nassiping Reforestation Project 67 50 28 Tree species diversity and structure of selected government reforestation projects 87

Quantitative Structure

A. Paraiso Reforestation Project

The density of trees was estimated at 993 trees/ha. The most dominant species was Swietenia macrophylla with 578 trees/ha followed by Reutealis trisperma (157 trees/ha), Tectona grandis (68 trees/ha), Adenanthera intermedia Merr. (27 trees/ha), Diospyros philosantera (24 trees/ha), and Pterocarpus indicus forma indicus (18 trees/ ha). These species were part of the original plantation species during the establishment of the reforestation project.

The basal area (dominance) occupied by all trees in the study area was computed at 40.66 sq m/ha. About 92% or 37.46 sq m/ha of the total dominance value was contributed by nine species only.

The average DBH of all trees was 18.4 cm and average tree height was 12.3 m. The maximum frequency was recorded for S. macrophylla, A. intermedia and D. philosanthera followed by R. trisperma and P. indicus. The dominance values of all species ranged from 0.01 to 17.24 sq m, the highest dominance of which was recorded for S. macrophylla.

S. macrophylla also showed the highest importance value (IV) index among the trees in all plots with 106.19% followed by R. trisperma (47.79%) and T. grandis (24.32). Other species had importance values of less than 10%. This showed that though there were many species of trees growing in this project area, S. macrophylla was the dominant species.

B. Nassiping Reforestation Project

Density ranged from 0.3 to 201 trees/ha, with T. grandis exhibiting the maximum density, while 22 other species recorded the minimum density.

The highest frequency was recorded for Ficus nota (Blanco) Merr. followed by T. grandis and lowest was recorded for the other 33 species. Among the plantation species, T. grandis exhibited the highest frequency followed by S. macrophylla and P. indicus forma indicus, Vitex parviflora and Gmelina arborea.

About 29.44 sq m or 92% of basal area was attributed to the major plantation species, of which S. macrophylla had the largest basal area of 9.36 sq m. The least dominant species in terms of basal area were Samanea saman (Jacq.) Merr., Mallotus philippinensis (Lam) Muell., and Pometia pinnata Forst & Forst. Trees with high 88 Sy et al. importance values were represented by teak (65.40%), mahogany (45.55%), yemane (25.87%), molave (23.72%), and narra (21.03%).

C. Marinduque Reforestation Project

Like in PRP and NRP, stand structure values in MRP were observed to be very high as well. For instance, S. macrophylla (168 trees/ha) exhibited high density followed by P. indicus (68 trees/ha) while V. parviflora (4 trees/ha) exhibited the minimum value among the plantation species. For the associated species, Triplaris cumingiana Fisch. & Mey recorded the highest density with 34 trees/ha followed by Macaranga tanarius (L.) Muell.-Arg with 33 trees/ha and Hevea brasiliensis (HBK.) Muell.-Arg with 23 trees/ha. As such, the density of all trees recorded ranged from 0.2 to 168 trees/ha.

The highest frequency among plantation species was recorded for P. indicus forma indicus followed by S. macrophylla and V. parviflora while M. tanarius exhibited the highest frequency among the associated species.

S. macrophylla emerged as the most dominant species in all plots with basal cover of 20.43 sq m among plantation species followed by P. indicus forma indicus with 9.10 sq m and V. parviflora with only 0.61 sq m. Intsia bijuga was the most dominant (1.57 sq m) among the associated species recorded in NRP.

Maximum importance value in NRP was recorded for S. macrophylla (90.80%) followed by P. indicus (42.67%), M. tanarius (11.26%) and lowest (0.35%) for 21 associated species.

Collectively, the plantation species represented majority of the total number of the recorded individuals per hectare. The composition of the three reforestation projects showed a high number of species represented by only few individuals. This indicates high diversity but with low species predominance. Such characteristic conforms to that of a tropical rainforest, where the number of species per unit area is particularly large and almost all species are represented by sparse population. According to Fedorov (1966), population of most species as a rule is extremely low in tropical rainforests.

Comparison of the three reforestation projects showed that the highest tree stand density was recorded in DBH class 10 (5 cm - 14.9 cm) and lowest in DBHclass >60 cm as shown in Figure 8. Tree stand density in the three reforestation sites consistently decreased with increasing DBH class. The trend conformed to a reverse “J” shape curve. Usually this type of diameter distribution is possessed by uneven- aged stand forest (Nyland 1996). Tree species diversity and structure of selected government reforestation projects 89

PRP NRP MRP T rees per hectare

Diameter class (cm)

Figure 8. Frequency distribution of trees in three reforestation projects.

Although the three sites were established as reforestation/plantation forest, tree species may not have been planted all at the same time resulting in uneven age distribution. Even if the trees were planted at the same time, with little or no management practices applied, mortality and regeneration of species would occur due to natural occurrence of canopy gaps. Uneven-aged stands eventually become dominated by shade tolerant species, because stand openings usually are too small to permit regeneration of intolerant species. Species that become established, persist, and grow reasonably well as advance regeneration, often fill the lower part of a diameter distribution (Smith 1986).

Species with higher importance value had greater contribution to the overall structure of the community. A tree species may have a higher share in determining the structure of the community on the basis of its density, dominance, or frequency of occurrence. However, when these attributes are combined, they may have lesser contribution to the community with respect to other species (Nyland 1996).

Not all organisms in the community are equally important in determining the nature and function of the whole community. Out of the great number of species present, a relatively few species generally exert major controlling influence by virtue of number, size, and other attributes. This does not mean that rare or less common species are not important; they are because they determine diversity, an important aspect of community structure (Odum 1971). This is true in the case of S. macrophylla as observed in the three reforestation projects. 90 Sy et al.

Based on stand density and basal cover, S. macrophylla was either dominant or co-dominant species in the three reforestation sites. This species is found in all forest types and it can adapt to a variety of soils but has a distinct preference for well- drained, sandy clay slopes. Young S. macrophylla trees are fairly tolerant to shade, but conditions such as canopy gap provide an opportunity for optimum growth. Since this was used as a pioneer reforestation species, its proliferation is inevitable given the suitable environmental conditions for growth.

Diversity Index

the diversity indices of the PRP, NRP, and MRP were computed at 2.77, 3.01, and 3.13, respectively. By comparison, the values of diversity index ranged from moderate to high based on modified Fernando Biodiversity Scale (1998) for the broad-leaved species in Mt. Makiling (Table 1). However, the result obtained in this assessment does not indicate similarity in species composition observed in Mt. Makiling. This index measures the number of species (species richness) in the community and their evenness in abundance. The diversity index is generally higher in tropical forests, with some reported as high as 5.06 and 5.40 for young and old stands, respectively (Knight 1975).

Equitability index (J) in the study sites as shown in Table 3 ranges from 0.58 to 0.72. NRP has a more even species distribution than PRP and MRP. MRP has the least even species distribution among the three study reforestation projects.

Table 3. Summary of diversity indices of the three reforestation projects. Diversity Index PRP NRP MRP Shannon-Weiner (H’) 2.81 3.04 2.59 Evenness (J) 0.65 0.72 0.58 Simpson’s Index (C) 0.84 0.91 0.85

The highest value for Simpson’s index of diversity was recorded in NRP (C=0.91), followed by MRP (C=0.85) and PRP (C=0.84). These values indicate high diversity within each project site. Thus, the probability that two randomly selected individuals in any of the plots established will belong to a different species. The computed results for the Shannon evenness index showed that there is relatively equal proportion of species in the three reforestation projects but the recorded data says otherwise. This is mainly influenced by the presence of large number of dominant species and few individuals for the less common species. Tree species diversity and structure of selected government reforestation projects 91

Simpson’s index is influenced by two parameters: 1) equitability of percent of each species present and 2) richness. For a given species richness, the index will decrease as the percent of each species becomes more equitable or the distribution of the number of individuals per species are almost equal. In the case of the three reforestation projects, only few species are represented by large population and majority of the species observed were rare or less common in terms of number.

the values for diversity indices (Shannon-Wiener and Simpson’s Diversity Index) conform to the study of Knight (1975), where diversity values in tropical forests are generally higher. Species diversity and richness are inherent characteristics of a tropical rainforest due to the favorable climate in the tropics and the long uninterrupted evolutionary history (Cain and Castro 1959). Higher diversity means longer food chain, more cases of symbiosis, and greater possibilities for negative feedback control which reduces oscillations and hence increases stability (Odum 1971).

Endemic, indigenous, and introduced species

A listing of all species observed in the three reforestation projects showed 11 common species out of the collective total of 165 species. These species are believed to be distributed throughout the Philippine archipelago.

Majority of the species observed in the three reforestation projects were indigenous and endemic species. Out of the 165 species, 101 were indigenous, 38 endemic, and 27 were introduced or exotic species (Table 5). The most common indigenous species among the three reforestation projects were Mallotus multiglandulosus, Lagerstroemia speciosa, Alstonia scholaris, Pterocarpus indicus, Litsea glutinosa, and Ficus nota.

Table 5. Number of endemic, indigenous, and introduced (exotic) species in the three study areas. Project area Endemic Indigenous Introduced or Exotic PRP 17 49 9 NRP 12 45 11 MRP 22 52 17

Based on the number of individuals, introduced species were recorded to dominate the three reforestation projects (Figure 9). Fast-growing species have the tendency to dominate and suppress the growth of other species promoting bio- invasion (Futuyma 1986 and Simberloff 1997 as cited by Gevaña and Pampolina 2007). However, the presence of endemic and indigenous species may suggest that the forests under these reforestation projects may not have been totally invaded by 92 Sy et al. introduced or exotic species. Given the favorable biophysical conditions, bio-invasion may be possible in this scenario. Therefore, it is wise that conservation management should be planned and implemented to protect biodiversity from the negative impacts of introducing exotic species.

Conservation status

A total of 21 species recorded in the three study sites were listed in the National List of Threatened Philippine Plants. Of this number, four species are Critically Endangered, six species are Endangered, seven species are Vulnerable, and four species are listed as Other Threatened Species as defined in the last section of the Methodology based on DAO 2007-01. Most of the species listed are timber- producing trees and economically important for furniture and high quality lumber. The presence of threatened species in each of the reforestation site studied also highlights the importance of these areas in biodiversity conservation as they house plants that are otherwise considered threatened in the wild. The list of species under critical biodiversity conservation is shown in Table 6.

700 600 500 400 Endemic 300 Indigenous 200 Introduced

N umber of individuals/ha 100 0 PRP NRP MRP Figure 9. Number and distribution of species according to species category. Tree species diversity and structure of selected government reforestation projects 93

Table 6. Species recorded in the three reforestation projects which are listed in the National List of Threatened Philippine Plants.

Species Common name Location Conservation status Adenanthera Tanglin Paraiso Other threatened intermedia species Afzelia rhomboidea Tindalo Paraiso, Endangered (Blanco) Vidal Nasiping Canarium Piling-liitan Paraiso, Nassiping, Other threatened luzonicum (Blume) Marinduque species A. Gray Canarium ovatum Pili Marinduque Other threatened Engl. species Cryptocarya ampla Bagarilao Paraiso Vulnerable Merr. Diospyros Bolong-eta Paraiso, Nassiping Endangered philosanthera Dracontomelon Dao Nassiping Vulnerable dao Blanco Merr. & Rolfe Intsia bijuga Ipil Paraiso, Marinduque Endangered (Colebr.) O. Kuntze Koordersiodendron Amugis Nassiping Vulnerable pinnatum (Blanco) Merr. Mangifera altissima Pahutan Paraiso, Marinduque Vulnerable (Blanco) Myristica Duguan Marinduque Other threatened philippensis (Lam.) species Palaquium Nato Paraiso, Marinduque Vulnerable luzoniense (F.-Vill.) Vidal Pterocarpus indicus Smooth narra Paraiso, Nassiping, Critically forma indicus Marinduque endangered Reutealis trisperma Baguilumbang Paraiso Critically endangered 94 Sy et al.

Table 6. Continuation Species Common name Location Conservation status Shorea negrosensis Red lauan Marinduque Vulnerable Foxw. Shorea polysperma Tangile Marinduque Vulnerable (Blanco) Merr. Sindora supa Merr. Supa Paraiso Endangered Syzygium nitidum Makaasim Paraiso, Marinduque Critically (Benth.) endangered Toona calantas Kalantas Marinduque Critically Merr. & Rolfe endangered Vitex parviflora Molave Nassiping, Endangered Marinduque Wallaceodendron Banuyo Paraiso Endangered celebicum Koord.

Conclusion

A total of 49 families, 120 genera, and 165 species were recorded in the three reforestation projects. Among the taxa, 38 species were found endemic, 101 species indigenous and 27 species were introduced. Most of the introduced species were economically important for furniture making, for light construction, and for ornamental purposes. Species such as Lagerstroemia speciosa (L.) Pers., Alstonia scholaris, S. macrophylla, P. indicus, and Canarium luzonicum were common among the three reforestation projects.

the highest density was recorded in PRP with species concentration in the 5-14.9 cm DBH. This consisted mostly of S. macrophylla which was found to be either dominant or co-dominant species in the three reforestation sites. The abundance of S. macrophylla in the three project sites indicates successful reforestation through the introduction of fast-growing species. However, this scenario poses a threat to local diversity as it was found that S. macrophylla has a potential for bio-invasion by limiting the growth of other species (Gevaña and Pampolina 2007).

High diversity values were noted for the three reforestation projects. This indicates that the area originally planted with two to five species had developed into multi-species through time. This can be attested by the observations in the three Tree species diversity and structure of selected government reforestation projects 95 reforestation areas. In the Paraiso Reforestation Project (PRP), established in 1930, in addition to the five major plantation species, 70 other tree species (belonging to 27 plant families) were added in its area in Barangay Tangaoan, Piddig, Ilocos Norte.

Second, the Nassiping Reforestation Project (NRP), established in 1939, had also flourished from the five major plantation species to a diversified stand consisting of 63 other tree species under 29 plant families.

Lastly, in Marinduque Reforestation Project (MRP) which was established in 1937, the area indicated the most number of species composition with 91 (including the major plantation species: narra and mahogany) under 36 different plant families.

Conservation status of species in the three reforestation projects showed a good number of native species. Of the 165 species listed for the three reforestation projects, 101 were indigenous, 38 were endemic, and 27 were introduced in the Philippines. Establishment of native species inside the reforestation project sites could have been the result of natural succession with the aid of different dispersal agents such as wind, wildfire, water (rainfall/typhoon), or human activities. From the native species observed, 21 were included in the National List of Threatened Philippine Plants (DAO 2007-01).

Recommendations

Although there are many species of trees growing in these forests, plantation or reforestation (mostly introduced) species were the dominant component and other species are only associated and still need to be established, especially for the indigenous and endemic species. Therefore, an integrated and intensive resource management in the three reforestation projects is needed to ensure the protection of biodiversity from the negative consequences of introducing exotic species. Further, the diversity and trend may serve as a relevant basis to determine the floristic hot spots that may contain threatened and endangered plant/tree species.

These reforestation projects contain possible sources of good or even superior planting materials for the different reforestation activities of the government and other organizations. Most of the species identified were indigenous and endemic to the Philippines which can be used in reforestation and forest rehabilitation activities. An effective biodiversity conservation management program, which includes protection, conservation and monitoring activities, should be planned and implemented to address diversity issues and reestablish species that were categorized as threatened, vulnerable, or endangered. 96 Sy et al.

Future conservation efforts should address the broad socio-ecological processes that are most likely to occur. The network of protected areas should be functionally integrated in a conservation strategy. In this context, study on impact of forest area on different community parameters will provide significant support to conservation planning by producing accurate information regarding local threats.

Lastly, there is a need for an increase in the level of awareness of the local government and the communities nearby on critical flora resources and plant diversity through the production and distribution of easily understandable Information, Education and Communication Campaign (IEC). This will help in encouraging the appreciation of forest diversity, especially if the area is being promoted as an ecotourism spot.

Literature cited

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Fernando ES. 1998. Forest formations and flora of the Philippines: FBS 21. College, Laguna: College of Forestry and Natural Resources, University of the Philippines Los Baños.

Futuyma D. 1986. Evolutionary biology. 2nd ed. Sinauer Associates. Sunderland, MA. Pp 3-4

Gevaña DT, Pampolina NM. 2007. Broadleaf diversity and carbon estimates along the northeastern slope of Mt. Makiling, Philippines. Sylvatrop Tech. J. Phil. Ecosystems and Nat. Res. 17 (1-2): 35-52.

[IUCN] International Union on Conservation of Nature and Natural Resources. 2001. IUCN Red list categories and criteria: Version 3.1. IUCN Species Survival Commission. IUCN, Gland, Switzerland and Cambridge, UK. Ii + 30p.

[IUCN] International Union on Conservation of Nature and Natural Resources. 2010. IUCN Red list of threatened species. Version 2010.4. [internet] [cited on15 November 2010] Available from: www.iucnredlist.org

Kent M, Coker P. 1992. Vegetation description and analysis: a practical approach. Boca Raton: CRC Press ; London: Belhaven Press, x, 363 p. Tree species diversity and structure of selected government reforestation projects 97

Nyland RD. 1996. Silviculture concepts and applications. New York: McGraw-Hill Co., Inc. 633 p.

Odum E. 1971. Fundamentals of ecology. 3rd ed. London: London Press.

Robert WP. 1974. An introduction to quantitative ecology. International student edition. McGraw-Hill series in population biology.

Rojo JP. 1999. Revised lexicon of Philippine trees. College, Laguna: Forest Products Research and Development Institute. p. 308.

Simberloff D. 1997. Strategies in paradise: Impacts and management of non-indigenous species in Florida. Washington DC: Istand Press. p 244-246.

Smith DM. 1986. The practice of silviculture. New York: John Wiley & Sons, Inc. 527 p.

Vermudo J. 1995. Vegetation analysis of the tree layer of lowland dipterocarp forest at Quezon National Park, Quezon Province, Philippines. [thesis]. Los Baños, Laguna: University of the Philippines Los Baños. 98 Sy et al.

Appendix Table 1. Summary of Species in Paraiso Reforestation Project. Species/Taxa Common Name Family Adenanthera intermedia Merr. Tanglin Fabaceae Afzelia rhomboidea (Blanco) Vidal Tindalo Fabaceae Albizia acle (Blanco) Merr. Akle Fabaceae Albizia lebbekoides (DC.) Benth Kariskis Fabaceae Alstonia scholaris (L.) R.Br. var. scholaris Dita Apocynaceae Anthocephalus chinensis (Lamk.) A. Rich. Kaatoan-bangkal Rubiaceae Antidesma bunius (L.) Spreng Bignai Euphorbiaceae Antidesma pentandum (Blanco) Merr. var. Bignai-pugo Euphorbiaceae pentandum Ardisia darlingii Merr. Malinasag Mysinaceae Arenga pinnata (Wurmb) Merr. Kaong Arecaceae Artocarpus nitidus Trec. ssp. nitidus Kubi Moraceae Broussonettia luzonica var. luzonica Himbabao Moraceae Buchanania arborescens (Blume) Blume Balinghasai Anacardiaceae Calophyllum inophyllum L. Bitaog Clusiaceae Canarium asperum Benth. ssp. asperum Pagsahingin Burseraceae Canarium luzonicum (Blume) A. Gray Piling-liitan Burseraceae Caryota cumingii Lodd. Pugahan Arecaceae Chisocheton cumingianus ssp. cumingianus Balukanag Meliaceae Cleisthanthus pilosus C.B. Rob. Banitlong Phyllanthaceae Cryptocarya ampla Merr. Bagarilao Lauraceae Cynometra ramiflora L. var. ramiflora Balitbitan Fabaceae Dehaasia incrassate (Blume) Leenh. Margapali Lauraceae Dimocarpus fumatus ssp. philippinensis Bulala Sapindaceae Dimocarpus longan Lour. ssp. malesianus Alupag Sapindaceae var. malesianus Diospyros philosanthera Blanco var. philo- Bolong-eta Ebenaceae santhera Ficus ampelas Burm. f. var. ampelas Upling-gubat Moraceae Ficus benjamina L. var. benjamina Salisi Moraceae Ficus cumingii Miq. var. terminalifolia Isis-kinis Moraceae Tree species diversity and structure of selected government reforestation projects 99

Appendix Table 1. Continuation

Species/Taxa Common Name Family Ficus irisana Elmer var. irisana Aplas Moraceae Ficus nota (Blanco) Merr. Tibig Moraceae Ficus variegata Blume var. variegata Tangisang-bay- Moraceae awak Intsia bijuga (Colebr.) O. Kuntze Ipil Fabaceae Kibatalia gitingensis (Elmer) Woods. Laneteng-gubat Apocynaceae Lagerstroemia speciosa (L.) Pers. Banaba Lythraceae Lepidopetalum perrottettii (Cambess.) Dapil Sapindaceae Blume Leucaena leucocephala (Lam.) de Wit Ipil-ipil Fabaceae Litsea glutinosa (Lour.) C.B. Rob. Sablot Lauraceae Mallotus philippensis (Lam.) Muell.-Arg. Banato Euphorbiaceae Mangifera altissima Blanco Pahutan Anacardiaceae Mangifera indica L. Mangga Anacardiaceae Melanolepis multiglandulosa (Reinw. ex Alim Euphorbiaceae Blume) Reichb. & Zoll. Milletia merrillii Perk. Balok Fabaceae Nauclea orientalis (L.) L. Bangkal Rubiaceae Nephelium lappaceum var. pallens (Hiem) Kapulasan Sapindaceae Leenh. Oroxylon indicum (L.) Kurz Pinka-pinkahan Bignoniaceae Palaquium luzoniense (F. Vill.) Vidal Nato Sapotaceae Parkia timoriana (DC.) Merr. Kupang Fabaceae Polyscias nodosa (Blume) Seem. Malapapaya Araliaceae Pongamia pinnata (L.) Merr. Bani Fabaceae Pterocarpus indicus forma indicus Smooth Narra Fabaceae Reutealis trisperma Baguilumbang Euphorbiaceae Samanea saman (Jacq.) Merr. Rain tree Fabaceae Sapindus saponarea L. Kusibeng Sapindaceae Semecarpus cuneiformis Blanco Ligas Anacardiaceae Semecarpus philippinensis Kamiring Anacardiaceae 100 Sy et al.

Appendix Table 1. Continuation

Species/Taxa Common Name Family Severinia retusa Malarayap-kutab Rutaceae Shorea assamica Dryer spp. philippinensis Manggasinoro Dipterocar- (Brandis) Sym. paceae Sindora supa Merr. Supa Fabaceae Spondias mombin L. Hogplum Anacardiaceae Sterculia cordata Blume var. montana Tapinag-bundok Sterculiaceae Sterculia foetida L. Kalumpang Sterculiaceae Swietenia macrophylla King Big-leaf Mahogany Meliaceae Syzygium calubcob (C.B Rob.) Merr. Kalubkub Myrtaceae Syzygium nitidum Benth. Makaasim Myrtaceae Syzygium subcaudatum (Merr. ) Merr. Malaruhat-bundok Myrtaceae Tamarindus indica L. Sampalok Fabaceae Tectona grandis L. f. Teak Lamiaceae Terminalia calamanasanai (Blanco) Rolfe Malakalumpit Combretaceae Terminalia microcarpa Decne. Kalumpit Combretaceae Terminalia nitens Presl Sakat Combretaceae Wallaceodendron celebicum Koord. Banuyo Fabaceae Wrightia pubescens R. Br. ssp. Laniti Lanete Apocynaceae (Blanco) Ngan

Appendix Table 2. Summary of Species in Nassiping Reforestation Project.

Species Common Name Family Afzelia rhomboidea Tindalo Fabaceae Aglaia elliptica Blume Malasaging-liitan Meliaceae Ailanthus integrifolia Lamk. ssp. integrifolia Malasapsap Simaroubaceae Alangium javanicum (Blume) Wang. Putian Cornaceae Alstonia scholaris Dita Apocynaceae Antidesma pentandum (Blanco) Merr. var. Bignai-pugo Euphorbiaceae pentandum Artocarpus blancoi (Elmer) Merr.. Antipolo Moraceae Artocarpus heterophyllus Lamk. Nangka Moraceae Tree species diversity and structure of selected government reforestation projects 101

Appendix Table 2. . Continuation

Species Common Name Family Artocarpus odoratissimus Blanco Marang-banguhan Moraceae Artocarpus ovatus Blanco Anubing Moraceae Averrhoa bilimbi L. Kamias Oxalidaceae Azadirachta indica A. Juss. Neem Anacardiaceae Bauhinia monandra Kurz Fringon Fabaceae Calophyllum blancoi Pl. & Tr. Bitanghol Clusiaceae Calophyllum inophyllum Bitaog Clusiaceae Canarium asperum Pagsahingin Burseraceae Canarium luzonicum Piling-liitan Burseraceae Chrysophyllum cainito L. Kaimito Sapotaceae Colona mollis (Warb.) Burr. Kedding Tiliaceae Diospyros philosanthera Bolong-eta Ebenaceae Dracontomelon dao (Blanco) Merr. & Rolfe Dao Anacardiaceae Evodia confusa Bugawak Rutaceae Ficus benjamina Salisi Moraceae Ficus congesta Roxb. var. melinocarpa Malatibig Moraceae Ficus irisana Aplas Moraceae Ficus melinocarpa Upli Moraceae Ficus nota Tibig Moraceae Ficus pseudopalma Niyog-niyogan Moraceae Ficus septica Burm. f. var. septica Hawili Moraceae Ficus ulmifolia Lam. Is-is Moraceae Ficus variegata Tangisang-bayawak Moraceae Gmelina arborea Roxb. Yemane Lamiaceae Heritiera littoralis Ait. Dungon-late Malvaceae Heritiera sylvatica Vidal Dungon Malvaceae Koordersiodendron pinnatum (Blanco) Amugis Anacardiaceae Merr. Lagerstroemia speciosa Banaba Lythraceae Leea guineensis G. Don Mali-mali Leeaceae 102 Sy et al.

Appendix Table 2. Continuation Species Common Name Family Leucaena leucocephala Ipil-ipil Fabaceae Litsea glutinosa Sablot Lauraceae Macaranga grandifolia (Blanco) Merr. Takip-asin Euphorbiaceae Macaranga tanarius (L.) Muell.-Arg. Binunga Euphorbiaceae Melanolepis multiglandulosa Alim Euphorbiaceae Mallotus philippinensis Banato Euphorbiaceae Mangifera indica Mangga Anacardiaceae Melicope triphylla (Lam.) Merr. Matang-araw Rutaceae Oroxylon indicum Pinka-pinkahan Bignoniaceae Palaquium foxwothyi Merr. Tagatoi Sapotaceae Parkia roxburghii Kupang Fabaceae Polyscias nodosa Malapapaya Araliaceae Pometia pinnata Forst. & Forst. Malugai Sapindaceae Pouteria campechiana Tiesa Sapotaceae Premna adenosticta Kalangiyawan Lamiaceae Psidium guajava L. Guava Myrtaceae Pterocarpus indicus forma indicus Smooth Narra Fabaceae Samanea saman (Jacq) Merr. Rain tree Fabaceae Sandoricum koetjape (Burm. f.) Merr. Santol Meliaceae Semecarpus cuneiformis Ligas Anacardiaceae Gomphandra luzoniensis (Merr.) Merr. Mabunot Icacinaceae Sterculia oblongata R. Br. Malabuho Sterculiaceae Streblus asper Kalios Moraceae Swietenia macrophylla Big-leaf Mahogany Meliaceae Symplocos odoratissima (Blume) Agosip Symplocaceae Tectona grandis Teak Lamiaceae Terminalia catappa L. Talisai Combretaceae Trichospermum involucratum (Merr.) Elmer Langosig Tiliaceae Vitex parviflora Molave Lamiaceae Vitex turczaninowii Merr. Lingo-lingo Lamiaceae Tree species diversity and structure of selected government reforestation projects 103

Appendix Table 3. Summary of Species in Marinduque Reforestation Project. Species Common Name Family Ailanthus integrifolia Malasapsap Simaroubaceae Albizia acle (Blanco) Merr. Akle Fabaceae Alphonsea arborea Bolon Annonaceae Alstonia scholaris Dita Apocynaceae Antiaris toxicaria (Pers.) Lesch. var. Upas Moraceae macrophylla Araucaria columnaris Araucaria Araucariaceae Areca catechu L. Bunga Arecaceae Artocarpus blancoi Antipolo Moraceae Artocarpus communis J.R. & G. Forst. Kamansi Moraceae Artocarpus heterophylla Nangka Moraceae Artocarpus ovatus Anubing Moraceae Bauhinia monandra Fringon Fabaceae Canarium luzonicum Piling-liitan Burseraceae Canarium ovatum Pili Burseraceae Cassia fistula Golden shower Fabaceae Cassia spectabilis Anchoan dilaw Fabaceae Casuarina equisitifolia Agoho Casuarinaceae Celtis luzonica Warb. Magabuyo Ulmaceae Celtis philippensis Blanco var. philip- Malaikmo Ulmaceae pensis Coffea arabica Coffee Rubiaceae Cordia dichotoma G. Forst Anonang Boraginaceae Delonix regia (Hook.) Raf. Fire tree Fabaceae Duabanga moluccana Blume Loktob Lythraceae Dysoxylum gaudichaudianum (A. Juss.) Miq. Igyo Meliaceae Endospermum peltatum Merr. Gubas Euphorbiaceae Enterolobium cyclocarpum (Jacq.) Earpod Fabaceae Griseb. Ervatamia pandacaqui Pandakaki Apocynaceae Erythrina orientalis (L.) Murr. Dapdap Fabaceae Evodia confusa Bugawak Rutaceae 104 Sy et al.

Appendix Table 3. Summary of Species in Marinduque Reforestation Project. Species Common Name Family Ficus ampelas Upling-gubat Moraceae Ficus callosa Willd. Kalukoi Moraceae Ficus congesta Malatibig Moraceae Ficus irisana Aplas Moraceae Ficus melinocarpa Upli Moraceae Ficus nota Tibig Moraceae Ficus septica Hawili Moraceae Ficus variegata Tangisang-bayawak Moraceae Gymnacranthera paniculata (A. DC.) Anuping Mysristicaceae Schout. Hevea brasiliensis (HBr.) Muell.-Arg. Para rubber Euphorbiaceae Hydnocarpus alcalae C. DC. Dudua Achariaceae Intsia bijuga Ipil Fabaceae Kleinhovia hospita L. Tan-ag Sterculiaceae Lagerstroemia speciosa Banaba Lythraceae Dendrocnide luzonensis (Wedd.) Chew Lipa Urticaceae var luzonensis Leucaena leucocephala Ipil-ipil Fabaceae Litsea glutinosa Sablot Lauraceae Macaranga bicolor Muell.-Arg. Hamindang Euphorbiaceae Macaranga grandifolia Takip-asin Euphorbiaceae Macaranga tanarius Binunga Euphorbiaceae Melanolepis multiglandulosa Alim Euphorbiaceae Mangifera altissima Pahutan Anacardiaceae Mangifera indica Mangga Anacardiaceae Melicope triphylla Matang-araw Rutaceae Miliusa vidalii Takulaw Annonaceae Mitephora lanotan Lanutan Annonaceae Myristica philippensis Duguan Myristicaceae Neolitsea vidalii J. Sincl. Puso-puso Lauraceae Nephelium lappaceum var. pallens Kapulasan Sapindaceae Tree species diversity and structure of selected government reforestation projects 105

Appendix Table 3. Continuation. Species Common Name Family Palaquium luzoniense Nato Sapotaceae Pangium edule Reinw. Pangi Flacourtiaceae Parashorea malaanonan (Blanco) Merr. Bagtikan Dipterocarpaceae Parasponia rugosa Blume Hanagdong Ulmaceae Persea americana Mill. Avocado Lauraceae Piliostigma malabarica Roxb. Alibangbang Fabaceae Pisonia umbellifera (Forst.) Seem. Anuling Nyctaginaceae Polyalthia flava Merr. Yellow Lanutan Annonaceae Polyscias nodosa Malapapaya Araliaceae Pouteria macrantha White Nato Sapotaceae Premna nauseosa Mulawin-aso Lamiaceae Pterocarpus indicus forma indicus Smooth Narra Fabaceae Pterocymbium tinctorium Taluto Sterculiaceae Sandoricum koetjape Santol Meliaceae Shorea negrosensis Red lauan Dipterocarpaceae Shorea polysperma Tangile Dipterocarpaceae Shorea squamata Mayapis Dipterocarpaceae Stemonorus luzoniensis Mabunot Icacinaceae Sterculia cordata var. montana Tapinag-bundok Sterculiaceae Sterculia crassiramea Tapinag Sterculiaceae Swietenia macrophylla Big-leaf Mahogany Meliaceae Syzygium nitidum Makaasim Myrtaceae Syzygium xanthophyllum Malatampui Myrtaceae Tamarindus indica Sampalok Fabaceae Tectona grandis Teak Lamiaceae Terminalia catappa Talisai Combretaceae Toona calantas Kalantas Meliaceae Trema orientalis Anabiong Cannabaceae Triplaris cumingiana Palosanto Polygonaceae Vitex parviflora Molave Lamiaceae Voacanga globosa Bayag-usa Apocynaceae 106 Sy et al. Importance Value ( %) 106.19 47.79 24.32 9.37 9.40 Relative Dominance (%) 42.40 26.88 15.11 1.05 1.34 Relative Frequency (%) 5.61 5.14 2.34 5.61 5.61 Relative Density (%) 58.19 15.77 6.88 2.72 2.45 Dominance (sq. m/ha) 17.24 10.93 6.14 0.43 0.54 Density (Individuals/ha) 578.00 156.67 68.33 27.00 24.33 Species Swietenia macrophylla Reutealis trisperma Tectona grandis Adenanthera intermedia Diospyros philosanthera Appendix Table 4. Top five species with highest Importance Values in Paraiso Reforestation Project. Tree species diversity and structure of selected government reforestation projects 107 Value (%) Importance 65.40 45.55 25.87 23.72 21.03 Relative Dominance (%) 28.34 29.36 11.21 10.01 13.48 Relative Frequency (%) 5.23 4.07 2.91 3.49 4.07 Relative Density (%) 31.82 12.12 11.75 10.22 3.48 (sq. m./ha) Dominance 9.03 9.36 3.57 3.19 4.30 Density (Individuals/ha) 201.33 76.67 74.33 64.67 22.00 Species echinatus Tectona grandis Swietenia macrophylla Gmelina arborea Vitex parviflora Pterocarpus indicus forma Appendix Table 5. Top five species with highest Importance Values in Nassiping Reforestation Project. 108 Sy et al. Value (%) Importance 42.67 11.88 9.79 9.21 Relative Dominance (%) 22.85 0.65 3.95 0.55 Relative Frequency (%) 6.21 4.58 3.27 1.96 Relative Density (%) 13.61 6.66 2.57 6.70 (sq.m/ha) Dominance 9.10 0.26 1.57 0.22 Density (Individuals/ha) 68.00 33.26 12.84 33.47 Species echinatus Pterocarpus indicus forma Macaranga tanarius Intsia bijuga Triplaris cumingiana Appendix Table 6. Top five species with highest Importance Values in Marinduque Reforestation Project. Sylvatrop, The Technical Journal of Philippine Ecosystems and Natural Resources 23 (1 & 2): 109 - 130

Genetic diversity of Limuran (Calamus ornatus Blume var. philippinensis Becc.) populations from three sites in Luzon Island, Philippines using Random Amplified Polymorphic DNA (RAPD) markers

Maria Theresa A. Delos Reyes, Ph.D. Aida B. Lapis, Ph.D. Science Research Specialist Scientist I Ecosystems Research and Development Bureau College, Laguna Nenita M. Calinawan [email protected] Science Research Analyst

Gracetine D. Magpantay Myrricar Loren G. Berdos Biologist I Biologist I

Aimee G. Cagalawan Biologist I

Genetic variation within and among populations of Limuran (Calamus ornatus Blume var. philippinensis Becc.) from Bataan, Camarines Norte, and Quezon were determined using Random Amplified Polymorphic DNA (RAPD) analysis. From the 19 primers used, 11 were polymorphic where a total of 182 alleles were detected. The total genetic diversity among populations and mean genetic diversity within population values were 0.4003 and 0.3724, respectively. The computed Wright’s Fixation Index WFI (Fst) was deemed moderate (0.0698) and could imply that the populations are relatively distant from each other. The dendrogram generated from the observed polymorphisms showed that the Bataan population, which has the highest genetic diversity among the three, was grouped singly. Statistical analysis of the genetic diversity and population genetic structure showed that the three populations have moderate variation. Results of the study can be used in the maintenance and/or improvement of Limuran in the Philippines.

Keywords: Calamus ornatus Blume var. philippinensis Becc., Limuran, genetic diversity, RAPD 110 Delos Reyes et al. rattans are climbing palms that are mainly found in asian countries which include the Philippines, Borneo, Indonesia, and Thailand (Sunderland and Dransfield 2002). It has been used by farmers and common folks for livelihood and subsistence in the Philippines (Richman 2006). It is particularly used in basket making, construction of temporary bridges, floorings, and furniture. In some countries, roots and leaves are boiled and used for medicinal purposes. The resin, sometimes called “dragon’s blood”, could be used as wood dye such as those found in violins and is also used to treat various illnesses (Ministry of Trade of the Republic of Indonesia 2010). The species is also considered by the Philippine government as priority for reforestation (Baja-Lapis 2009).

Deforestation and the non-sustainable extraction of rattan from primary and secondary forests gave rise to serious concern on its future availability, specifically the loss of important genetic resources in the Philippines. The Department of Environment and Natural Resources (DENR) is aware of the critical situation of many rattan resources and has already initiated appropriate action to prevent further loss. Researchers of the Ecosystems Research and Development Bureau (ERDB), the principal research arm of the DENR, surveyed the current status of rattan resources in the Philippines by gathering relevant information on abundance and distribution of national priority species and collecting seeds for the establishment of plantations and ex-situ gene banks (Tesoro 2002). Fragmentary actions such as micro-propagation by tissue culture had also been initiated but the basic premise in doing all these endeavors has been left behind, that is, genetic structure/markers determination. Genetic variation, an important factor to consider in breeding, determines the species’ ability to adapt to changing environmental conditions over time, the species’ ability to occupy new ecological niches and the species’ fitness (Rao and Hodgkin 2002; Finkeldey 2005). There is a pressing need to evaluate the genetic diversity of existing rattan plantations to design strategies in maintaining and/or improving rattan.

Understanding the diversity of the elite lines requires both morphological and molecular assays. Several markers are available to test the genetic diversity of certain populations and among these is the Random Amplified Polymorphic DNA (RAPD). It uses a single “arbitrary” primer that randomly amplifies any part of the genome. This technique has been widely used in many genetic studies because of its high potential to detect polymorphisms despite being a dominant marker and its reproducibility problems. It is also very simple, inexpensive, and does not require complicated techniques to produce results (Kumar and Gurusubramanian 2011; Bardakci 2001).

This study aimed to assess the genetic diversity within and among populations of C. ornatus Blume var. philippinensis Becc. from existing germplasm sources in Bataan, Camarines Norte, and Quezon using RAPD markers. Genetic diversity of Limuran populations 111

Review of literature

Limuran (Calamus ornatus Blume var. philippinensis Becc.)

Calamus, the largest genera of Rattans, came from the Malay word “rotan” meaning “climbing palms”. It belongs to family Arecaceae and composes a total of 388 accepted species names including the Philippine rattans Calamus merrillii and Calamus ornatus Blume. Members of the genus are mostly sexually and asexually reproducing. They are usually found in Asian countries including Borneo, Indonesia, Philippines, and Thailand (The Plant List; Sunderland and Dransfield 2002). Two of the most commonly used species of rattan are found in the Philippines: Palasan (Calamus merrillii Becc.) and Limuran (C. ornatus Blume var. philippinensis Becc.).

C. ornatus Blume var. philippinensis Becc., commonly known as “limuran” is endemic to the Philippines, specifically in the lower slopes of Mount Makiling and low and medium altitudes of primary forests (De Guzman 1986). It is characterized by triangular, semi-ring spines that are diagonally arranged, with bulbous knee and alternate leaflets. It has sheaths that are dull green and densely spiny and stems which are 4-7 cm thick and can reach up to 20 m tall. Infructescence forms two to three clusters which are about 90-150 cm apart. The fruit, which contains only one 18-mm long brownish black oblong seed, is 30-35 mm long and 10 mm wide, ellipsoid or ovoid with distinct black margins (Baja-Lapis 2010).

Limuran canes are used for furniture, walking sticks, handles for implements and flooring; leaves, cabbage and roots as medicine while fruits are occasionally eaten (Baja-Lapis 2010). The fruits are juicy and are used to make vinegar because of the sour taste. The fruits are collected from the wild and taken to local markets during the fruiting season which runs from November to January. Limuran is not cultivated for fruit but planted widely and commercially for the production of cane. The young terminal shoots of limuran serve as food and medicine to the Aetas (Yu 2007). The pioneering study revealed rattan’s bioactive components and molecular structures that are anti-inflammatory and anti-diarrhetic (Yu 2007). Therefore, Calamus ornatus shoot is a potential functional food to help aid the cure of diarrhea and provide significant active anti-inflammatory components.

Because of its variety of uses, rattan has been studied from simple determination of morphological characteristics (Baja-Lapis 1983; Baja-Lapis 1997) to phylogenetic studies using intron and spacer sequence data (Baker et al. 2000a; Baker et al. 2000b). With this DNA sequence information, accompanied by datasets of morphological data, a new phylogenetic tree for rattan was generated (Dransfield et al. 2005). Researches on in vitro mass propagation of some species of Calamus, and 112 Delos Reyes et al. on phylogeny and taxonomy using molecular markers continued (Goh et al. 2001; Kumar et al. 2012a; Kumar et al. 2012b; Sreekumar et al. 2006). In the Philippines, efforts have been done on the assessment of genetic diversity of Palasan, Calamus merrillii Becc. (Delos Reyes et al. 2010; Delos Reyes et al. 2011b) and Limuran (Calamus ornatus Blume var. philippinensis Becc.) (Delos Reyes et al. 2011a) using isozyme analysis. The most recent study on Calamus is the identification of the psbA- trnH intergenic spacer locus (PTGIS) as the single barcode for species discrimination (Yang et al. 2012).

While other countries have moved at a fast pace with molecular studies on rattan, the Philippines, however, is still lagging behind in terms of programs centered in the development of rattan research (House Bill 6470 2012).

Random Amplified Polymorphic DNA (RAPD)

Molecular markers are considered as powerful tools in the field of molecular biology and biotechnology. They are commonly used to determine polymorphisms in the genome which can be used in evaluating genetic diversity within and among populations (Kumar et al. 2009). Hybrid character expression can be achieved through these markers by understanding the genetic basis of morphological differences between species (Riesberg and Ellstrand 1993); understanding the genetics of plant- pathogen interaction (Benali et al. 2011); as well as determining the correlation between pest and diseases (Berbegal et al. 2011).

Several types of molecular markers are available depending on the application being carried out. These are divided into two kinds: PCR-based and non-PCR-based markers. PCR-based markers include Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSRs), Simple Sequence Repeats (SSRs), and Single Nucleotide Polymorphisms (SNPs) (Mondini 2009). Non-PCR-based marker include RFLP/Restriction Fragment Length Polymorphism (Mondini 2009).

Though very informative, there is no molecular marker that is capable of generating all the necessary genetic information about a particular organism. They differ in the information that they provide, as well as the cost, level of equipment needed, and degree of technical expertise (Muchugi et al. 2008). Pertinent details regarding the information being sought, the nature of the sample being used, and the genetics of the species (genome, mode of reproduction, and distribution) are all considered before selecting a particular marker technology (McCartney et al. 2003). Genetic diversity of Limuran populations 113

The marker technology used in this study is RAPD. This marker uses short, random, 10 bases primers that attach randomly to complementary sites in the DNA (Muchigi et al. 2008). This marker is advantageous in cases when there is no available information regarding the genome or the genetic sequence of the organism to be studied. Despite its limited reproducibility and dominant nature, it is relatively simple and inexpensive compared to other marker technologies (Mondini 2009; Muchigi et al. 2008).

RAPD markers are commonly used to assess relationships and genetic diversity of organisms for understanding and remodeling phylogenetic trees, and for breeding programs (Sreekumar et al. 2006; Salwana et al 1998). The markers were used to determine genetic diversity as support to the merging of Calamus rivalis Thw. ex Trim and Calamus metzianus Schlecht as a single species (Sreekumar et al. 2006) and in the correct identification of Amaranthus species (Popa et al. 2010). They were also used to determine the relationship of the geographic location of Alternaria alternate to its genetic diversity and its virulence to citrus hybrids in Iran (Kakvan et al. 2012).

Genetic Diversity

Brown (1983) defined genetic diversity as the amount of genetic variability among individuals of a variety, or a population of a species. Genetic diversity data is important in taxonomy, conservation, origin and evolution studies. It can provide information on the level of variation, and clustering and relationships regarding the taxonomic groups of organisms which in turn can help researchers define the extent of their research. For instance, a population with low genetic diversity will not be a good choice as source of seeds for breeding because low variation will increase the chance of inbreeding which may lead to low seed yield, germination rate, and survival (Finkeldey 2005).

Genetic diversity also aids in the understanding of the origin and evolution of organisms which is important in selecting the secondary and tertiary gene pools in the study. Lastly, diversity data has an effect in the choice of organisms to be conserved and improved by determining the status of populations, therefore inferring the degree in which organisms are managed and used (Rao and Hodgkin 2002).

Statistics of genetic diversity and population genetic structure

Bias in estimating allele frequencies arises because of the assumption that populations always follow the Hardy-Weinberg equilibrium. But such is not commonly observed in nature. To eliminate the bias in estimating allele frequencies, the Bayesian method with non-uniform distribution is commonly used in diversity studies. Probabilities of Bayesian method are computed by combining the prior 114 Delos Reyes et al. probabilities with the data obtained (Kindt et al. 2009). Zhivotovsky (1999) developed two different methods of computing prior probabilities. One is the uniform prior distribution and the other is the non-uniform prior distribution. Bayesian computations based on non-uniform priors are said to provide the most reliable allele frequencies estimates (Kindt et al. 2009).

Aside from determination of allele frequencies, another test is usually performed to support diversity studies. Felsentein (1985) introduced the use of bootstrap to determine the confidence levels of evolutionary trees estimated from DNA or RNA data. Bootstrap, as described by Efron and Tibshirani (1993) is a computer- based technique for assessing the accuracy of almost any statistical estimate.

In Felsenteins’ bootstrap, a bootstrap matrix (x*) is generated from random selection of data from the original matrix (x). The columns of x may be rearranged in any order in x*. Random selection of data will proceed until x* is generated. Then, the tree building algorithm is applied to x* to generate the bootstrap tree (TREE) (Efron et al. 1996; Felsenstein 1985).

The process will be repeated B times (in this study, B = 10,000 steps). Then, the percentage of the bootstrap trees that conforms to the original tree is calculated. Similar trees have the same overall look regardless of the length of the branches (Efron et al. 1996). Bootstrap analyses are usually done in phylogenetic and diversity studies to understand and/or verify the relationships between taxa and/or populations. Some of the studies that used this method include genetic diversity analysis of crop plants (Mohammadi and Prasanna 2003); phylogenetic analysis which found evidence on the convergence between the “yeti”and primates (Milinkovitch et al. 2004); analysis on the molecular evolution of histone deacetylase (Gregoretti et al. 2004); RAPD and ISSR Analysis of 12 species and 3 subspecies of Grevillea (Pharmawati et al. 2004); verification of phylogeny and evolutionary relationships of genus and species of mosquitoes (Diptera: Culicidae) based on 18S DNA sequences (Shepard et al. 2006); molecular characterization of Solanum tuberosum L. using SSR and RAPD markers (Rocha et al. 2010); and genetic diversity analysis of Indian teak (Tectona grandis L.f.) populations using ISSR markers (Ansari et al. 2012).

Methodology

Place of implementation

The research was conducted at the ERDB Forest Molecular Laboratory, College, Laguna from January 2011 to June 2012. Genetic diversity of Limuran populations 115

Collection and DNA Extraction

Leaves of 30 plant samples of C. ornatus Blume var philippinensis Becc were collected from each of the following towns in Luzon island, Philippines: Abucay, Bataan (BAL); Daet, Camarines Norte (DAL); and Pagbilao, Quezon (QL). Young leaves were cut, ground with liquid nitrogen, and extracted using the modified extraction protocol by Pirttila et al. (2001). The DNA samples were normalized to 1ng/µL and diluted in a 1:50 dilution.

Amplification

Screening of the 19 RAPD primers for the three representative samples from each of the populations was performed. This was done to assess if the primers would work for Limuran. The primers used in this study were synthesized by Invitrogen. Primers with positive results were used to amplify all of the limuran samples (Table 1).

Table 1. List of RAPD primers that successfully amplified segments of the genome of Calamus ornatus Blume var. philippinensis Becc. collected from Bataan, Camarines Norte, and Quezon. Primer Sequence (5'-3') OPA03 AGTCAGCCAC OPA04 AATCGGGCTG OPA11 CAATCGCCGT OPA12 TCGGCGATAG OPA13 CAGCACCCAC OPA15 TTCCGAACCC OPA16 AGCCAGCGAA OPA17 GACCGCTTGT OPAU02 CCAACCCGCA OPAW07 AGCCCCCAAG OPAW09 ACTGGGTCGG

The protocol devised by Sreekumar et al. (2011) was modified to suit the problems encountered with Limuran. In the modified protocol, 25µ L reaction mixture contained the following components: 2.0 µL DNA, 1x PCR buffer with 1.5 mM MgCl2 (KAPA Biosystems), 1 µM RAPD primer, 0.2 mM dNTPs (KAPA Biosystems), and 0.2 µL (1.5 U) Taq Polymerase (KAPA Biosystems). Amplification was performed using Bio-Rad and Applied Biosystems Veriti thermal cyclers. Initial denaturation was 116 Delos Reyes et al. set at 94°C for seven minutes; followed by 45 cycles of the following: 1 minute denaturation at 94°C, 1 minute annealing at 36°C, and 2 minutes extension at 72°C. Final extension was set at 72°C for 10 minutes. Amplified products were visualized via electrophoresis (1.8% agarose, SYBR Safe Stain) and the resulting band patterns were documented using the BioRad EZ gel imager and the ImageLab software.

To overcome the problem of reproducibility of RAPD, amplification was performed twice or thrice and only bands present in all amplifications were scored. Resulting bands were scored as follows: 1- band is present; 0- band is absent. A binary matrix was generated for all populations. This was then converted into formats suitable for different genetic diversity programs using GenAlEx 6 (Genetic Analysis in Excel).

Generation of genetic variation within and among populations

Genetic variation within and among populations were computed using ALFP surv software. This software uses Bayesian estimation using non-uniform prior distribution of allele frequencies. This method was developed to minimize bias in frequency computations (Zhivotovsky 1999). The frequency of the null allele at each locus was computed from two values: sample size and number of individuals that lack the bands. The distribution of allele frequencies is then estimated separately for each population when several populations are tested (Vekemans et al. 2002).

After estimating the allele frequencies, the gene diversity and population genetic structures were computed. The genetic diversity has different components, namely: number of loci, proportion of polymorphic loci (PLP), expected heterozygosity or Nei’s genetic diversity (Hj) and its variance components, and average gene diversity within populations or samples (Hw) and its variance components (Vekemans et al. 2002) — all of which were computed and tabulated.

The total number of loci is defined as the total number of monomorphic and polymorphic loci in a sample. IPGRI and Cornell University (2003) defined proportion of polymorphic loci (P or PLP) as the number of polymorphic loci (npj) divided by the total number of loci (ntotal) stated as follows:

Expected heterozygosity (Hj) is the probability that any two alleles, chosen at random from the population, are different from each other at a single locus (IPGRI and Cornell University 2003). Genetic diversity of Limuran populations 117

where Hj is the estimated expected heterozygosity, L is the total observed loci, and i as locus (Lynch and Milligan 1994).

Gene diversity within populations (Hw) was computed as follows:

where Hw is the estimated gene diversity within populations, n is the total number of samples, j is the locus, and Hj is the estimated expected heterozygosity (Hj) (Lynch and Milligan 1994).

Statistics of population genetic structure, on the other hand, include the following: total gene diversity (HT), average gene diversity within populations

(Hw), average gene diversity among populations in excess of that observed within populations (Hb), and Wright’s Fixation Index (Fst) (Vekemans et al. 2002).

Total gene diversity (HT) is defined as the sum of gene diversity within populations (IPGRI and Cornell University 2003). Average gene diversity among populations (Hb) was computed by averaging all distinct pairs of populations.

where Hb is an estimate of average gene diversity among populations in excess of that observed within populations, j and k as two distinct populations (Lynch and Milligan 1994).

The total gene diversity that occurs among as opposed to within populations or the Wright’s Fixation Index (Fst) is computed as follows:

where Fst is an estimate of Wright’s Fixation Index, Hb is an estimate of average gene diversity among populations in excess of that observed within populations, HT is the total gene diversity, Var(Hw) is the variance of the estimate of gene diversity within 2 populations, Cov(Hb,Hw) is the covariance Hb and Hw, and HT is the square of the total gene diversity (Lynch and Milligan 1994). 118 Delos Reyes et al.

The AFLP Surv (Vekemans 2002) software generates matrices that are used to create dendrograms using PHYLIP (Felsenstein 2009) software. The resulting dendrogram shows the relationship among limuran populations. A dendrogram showing the relationship of each individual in a population can be generated using DARwin 5.0 (Perrier et al. 2003; Perrier and Jacquemound-Collet 2006) software. AFLP Surv was also used to perform bootstrap analyses to support the dendograms generated. Bootstrap analyses (10,000 iterations) were performed.

Results and discussion

Screening of the 19 primers for three representative samples from each population was performed. Of the 19 primers, only 11 were able to amplify segments in the genome (Table 1).

Intrapopulation genetic diversity

The proportion of polymorphic loci (PLP) expresses the percentage of variable loci in a population (IPGRI and Cornell University 2003). PLP from both the Camarines Norte and Quezon populations were the same at 98.4% since both populations observed 179 polymorphic loci from a total of 182 loci (Table 2). On the other hand, all loci from Bataan showed polymorphism (Table 2). A high PLP value signifies that the alleles found in all Limuran populations are highly variable. A high PLP value for the other rattan species was also observed (Sreekumar et al. 2006).

Table 2. Intra-population genetic data of Calamus ornatus Blume var philippinensis Becc. populations collected from Bataan (BAL), Camarines Norte (DAL), and Quezon (QL). Number of 1 2 3 Population n No. of loci polymorphic PLP Hj loci Bataan 21 182 182 100.0% 0.37658

Camarines Norte 27 182 179 98.4% 0.36632

Quezon 30 182 179 98.4% 0.37419

Legend: 1 number of samples 2 proportion of polymorphic loci 3 expected heterozygosity Genetic diversity of Limuran populations 119

The computed Nei’s genetic diversity (Hj) values were 0.37658, 0.36632, and 0.37419 for Bataan, Camarines Norte, and Quezon, respectively which are higher than the values obtained in past genetic diversity studies of Calamus (Sreekumar 2006). This variation may be attributed to the species’ characteristics of being outcrossing and insect-pollinated (Sreekumar 2006) which led to higher probability of recombination (Finkeldey 2005)

Gene diversity

The total gene diversity (Ht) among all populations was high at 0.4003 which is higher than the value obtained for Coreopsis leavenworthii (0.309) (Czarnecki II et al. 2008) while the mean gene diversity within populations (0.3724) was higher than diversity values of other species of rattan, and other organisms: Calamus palustris – 0.153 (Isozyme) (Salwana et al. 1998), Calamus rivalis – 0.1793 (RAPD) (Sreekumar et al. 2006), Picea spp. – 0.27 RAPD (Narendrula and Nkongolo, 2012), Tylophora rotundifolia - 0.2643 (RAPD) (Sebastian et al. 2010); but lower than Dendrobium spp. - 0.5612 (RAPD) (Chattopadhyay et al. 2012). Relative to these values, the Limuran populations are moderate in genetic variability. The level of genetic diversity within and among populations determines the survival and adaptation of the individuals in a population (Neel and Ellstrand, 2003; Anand et al. 2004). A considerable amount of genetic diversity lowers the chance of inbreeding depression which may affect the organism’s yield and survival performance (Finkeldey 2005). Therefore, the conservation of the diversity of the Limuran populations is important in the survival and adaptation of the species over time (Catana et al. 2013).

The computed genetic differentiation value among populations (Hb) was described as the average gene diversity among populations in excess to that observed within populations. The genetic differentiation value was found to be moderate at 0.0279 which indicates a considerable amount of distinction among the three populations (Table 3).

Table 3. Gene diversity within and among Calamus ornatus Blume var philippinensis Becc. populations. 1 2 3 Ht Hw Hb Fst 0.400300 0.372400 0.027900 0.069800 S.E. 0.003098 0.000000 0.008463 Var. 0.000001 0.000000 0.000072 1 total gene diversity among populations 2 mean gene diversity within populations 3 genetic differentiation among populations 120 Delos Reyes et al.

Wright’s Fixation index (Fst) measures genetic correlation between pairs of samples within a population relative to pairs of samples obtained in the populations or can be interpreted as the total gene diversity that occurs among as opposed to within population (Vekemans 2002). The observed Fst for Limuran was moderate at 0.0698 (Table 3) which indicates that only 6.98% of variation exists among populations. This shows that there is more diversity to be found within populations than among the Limuran populations. This observation was also noted in a study by Sreekumar et al. (2006) in rattan using RAPDs.

The presence of gene diversity and differentiation within and among populations, respectively, may be attributed to cross pollination and differential selection pressures acting between populations like differences in geographic location especially the presence of geographic barriers like large bodies of water (Cardoso et al. 2000; Sebastian et al. 2010). The three populations studied are not separated by geographic barriers but are considerably distant from each other which could explain the moderate genetic differentiation value. This likelihood of the three populations to differentiate from each other may be because of possible chance of cross-pollination. This probability of cross pollination may have contributed as to why there is higher variation within populations than among populations.

Cluster analysis

To understand the genetic relationship among the three populations, a consensus tree was generated using the PHYLIP software. The tree shows that Daet (Camarines Norte), and Pagbilao (Quezon) populations grouped together to form a cluster, whereas Abucay (Bataan) was grouped singly. Bootstrap analysis confirmed that the generated tree has an acceptable bootstrap support of 65.94% (Figure 1) which is slightly lower than 70%, the threshold value of the trueness of a phylogenetic tree (Brinkman and Leipe 2001; Baldauf 2003).

The Bataan population formed a separate group from the other two in the consensus tree generated using PHYLIP software. This may be because Bataan is more geographically distant from the two locations compared to the distance between Camarines Norte and Quezon. This suggests that the Camarines Norte and Quezon populations interact more often possibly through seed exchange or dispersal or cross- pollination. However, with a bootstrap support value of 65.94%, moderate values of differentiation and fixation, and close values of genetic diversity within populations, it can be said that the three populations are probably interacting with each other at near rates. Genetic diversity of Limuran populations 121

BAL 1-30 DAL 1-30 QL 1-30

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1kb A 0.5 kb

BAL 1-30 DAL 1-30 QL 1-30

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Figure 1. Electrophoretogram of Limuran samples amplifi ed using (A) OPAU02 and (B) OPAW07 RAPD primers run in 1.8% agarose at 180V for 180 minutes.

Figure 2. Consensus tree of Calamus ornatus Blume var philippinensis Becc. populations sampled from Bataan (BAL), Camarines Norte (DAL), and Quezon (QL) generated from PHYLIP software with 10,000 bootstrap iterations.

Another dendrogram of C. ornatus Blume var philippinensis Becc. populations was generated from DARwin 5.0 software to further support the clustering information of the three populations (Figure 3). The tree, with a very low bootstrap support value of 2% shows that the samples from the Bataan, Camarines Norte, and Quezon populations clustered distinctly from each other. This means that there is only a 2% confi dence level that the three populations are distinct from each other. 122 Delos Reyes et al.

Figure 3. Dendrogram (vertical heirarchical tree) for the gene frequency of Calamus ornatus Blume var philippinensis Becc. populations sampled from Bataan (BAL), Camarines Norte (DAL), and Quezon (QL) generated using DARwin 5.0 software by Weighted Neighbour Joining using JACCARD coefficient. Numbers on the tree represent bootstrap analysis performed on the clusters.

Another dendrogram of C. ornatus Blume var philippinensis Becc. populations, was generated from DARwin 5.0 software to further support the clustering information of the three populations (Figure 3). The tree, with a very low bootstrap support value of 2% shows that the samples from the Bataan, Camarines Norte, and Quezon populations clustered distinctly from each other. This means that there is only a 2% confidence level that the three populations are distinct from each other. Genetic diversity of Limuran populations 123

Genetic distance

The genetic distance between Bataan and Camarines Norte populations was 0.0534 compared to Bataan vs. Quezon at 0.0437, and Camarines Norte and Quezon at 0.0395 (Table 4). Genetic distance determines the relation of populations from each other. Results of the genetic distance data showed that Camarines Norte and Quezon were the most genetically similar whereas Bataan and Camarines Norte were the most distant. This data supports the consensus tree generated using PHYLIP software wherein Camarines Norte and Quezon clustered together while Bataan separated.

Table 4. Genetic distance and Pairwise Fst between populations of Calamus ornatus Blume var philippinensis Becc. generated using the AFLP surv software in three sampling sites: Bataan, Camarines Norte, and Quezon. Populations of Limuran Genetic distance* Bataan vs. Camarines Norte 0.0534 Bataan vs. Quezon 0.0437 Camarines Norte vs. Quezon 0.0395 *Nei’s genetic distance (Lynch and Milligan,1994).

Conclusion

Genetic variation within and among populations of Calamus ornatus Blume var philippinensis Becc. from Bataan, Camarines Norte, and Quezon were determined using RAPD. From the 19 primers used, 11 were polymorphic with a total of 182 alleles detected. Results showed that total genetic diversity among populations and mean genetic diversity within population values were 0.4003 and 0.3724, respectively, showing presence of variation in the populations. Wright’s fixation index (Fst) was moderate at 0.0698. These imply that the three populations are relatively distant from each other. This genetic variation could be due to cross pollination or geographical differences.

The dendrogram generated from DARwin using the weighted neighbour (WNJ) joining algorithm showed that Bataan population has the highest genetic diversity among the three populations which could be due to its geographical separation from the two other sites.

Analysis of the genetic diversity and population genetic structure showed that the three populations have moderate genetic diversity and can serve as good sources of potentially useful genes. 124 Delos Reyes et al.

The assessment of the genetic diversity for the three populations of Limuran (Calamus ornatus Blume var philippinensis Becc.) proves that the individuals in the existing Limuran plantations in the Philippines are moderately variable.

Diverse populations are said to be more tolerant to changes in the environment since its genes are able to adapt quickly to environmental changes. Thus, the populations studied are sturdy enough to survive stresses from the environment and can serve as sources of potentially useful genes for breeding.

Another genetic diversity study should be conducted using more informative markers like Single Nucleotide Polymorphisms (SNPs) or Simple Sequence Repeats (SSR) to determine if there is gene flow to proceed to conservation.

Acknowledgment

The authors are grateful to the ERDB management for the continuous support of project funds and acquisition of equipment for the ERDB Forest Molecular Laboratory. Sincere thanks is also extended to the following: Dr. Maria Genaleen Q. Diaz, Associate Professor 5 and her staff, Mr. Wilson Aala, Jr., Genetics and Molecular Biology Division, Institute of Biological Sciences, College of Arts and Sciences, UPLB for sharing their time and expertise; Dr. Kenneth McNally (Head, Genetic Resources Center, International Rice Research Institute, College, Laguna) and staff, Mrs. Elizabeth Naredo and Ms. Sheila Quilloy for the accommodation and support; and to our Sovereign God the Father and Source of Everything, our highest praises, honor, and thanks for the wisdom and success of this endeavour.

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Sylvatrop, The Technical Journal of Philippine Ecosystems and Natural Resources 23 (1 & 2): 131 - 148

Expression of dominance as influenced by soil fertility in Imperata cylindrica- Saccharum spontaneum vegetation formation

Justino M. Quimio, Ph.D. Romeo S. Capon, Jr. Professor Science Research Assistant Visayas State University Baybay City, Leyte [email protected]

Kathy Jane S. Vergara Science Research Assistant

The study was a two-year experiment involving six 4 m x 4 m plots in mixed Imperata cylindrica and Saccharum spontaneum grassland formation, with application of 14-14-14 NPK fertilizer as the treatment. It aimed to determine which of the two grass species would express vegetative dominance over the other when soil gains in fertility.

Results showed that Saccharum spontaneum gained dominance when fertilized, with increase in height, plot cover, number of shoots, and biomass production and the consequent reduction in number of shoots and biomass production in Imperata cylindrica. The results supported the current grassland succession models that are based on field observation, which suggest that Imperata and Saccharum tend to occur in mixture in denuded sites but Saccharum tends to express dominance over Imperata in more productive sites. Frequent disturbance to biomass in Imperata- Saccharum formation, such as harvesting or by fire, can prevent Saccharum from attaining competitive dominance over Imperata. The consequence is for Imperata to maintain its dominance because of removal of competition pressure from potentially larger plants like the Saccharum, shrubs, juvenile trees, and associated persistent vines. This study recommends further studies

Keywords: species competition, grassland ecology, ecological succession 132 Quimio et al.

on the ecology of Philippine grasslands to establish basic information for more effective land management and site capability evaluation.

rehabilitation of denuded grasslandS remains to be in the forefront of forest resource conservation and ecological restoration. Much of the reforestation budget goes to fire prevention especially that most reforestation sites are dominated by the fire-prone grass,Imperata cylindrica. The concern on grassfire becomes worse when Imperata is mixed with Saccharum spontaneum, also a grass but harder to eradicate, and produces much higher amount of combustible material that cause more intense burning.

In a survey conducted by Quimio (1996) on grassland vegetation in Leyte which included samples dominated by Imperata but without Saccharum, Imperata mixed with Saccharum and pure stand of Saccharum, the Imperata grassland emerged as soil in cultivated forest lands became impoverished. Furthermore, with domination by Imperata, the land is abandoned to long cycles of regular burning. In years of regular burning, Saccharum can invade Imperata grassland and later may develop dominance on the land.

Because of node buds in its stubble, Saccharum can survive fire by producing new shoots after burning instead of starting from seed again to maintain presence in its place. Napiza (2000) reported the stubble of Saccharum as reaching depth of over one meter in well-drained soils of Leyte. Saccharum grows well in soils with low sodicity and absence of clay kankar pan throughout the root zone (Singh and Jha 1993). This deeply entrenched stubble makes Saccharum hard to eradicate. The possession of underground buds from which to grow after disturbance is a strategy common to Imperata and Saccharum in surviving grassfire. With the advantage of greater lateral spread and height than Imperata, Saccharum can out-compete Imperata in good soils or where productivity is adequate for both to attain optimum potential sizes.

There is a general understanding on improvement in soil structure and nutrient status when land is left to fallow, or as success in progresses. The grassland succession model developed by Quimio (1996) suggests that the ecological species group of Imperata cylindrica is being replaced by the ecological species group of Saccharum spontaneum after years of influence by regular burning. Thus, theoretically, it could be presumed that improvement to soil should have happened as vegetation shifts from one that is dominated by Imperata into one that is dominated by Saccharum.

The study of Jayme (1998) on Imperata-Saccharum vegetation gradient indicated better soil nutrient status at plots that show dominance by Saccharum Expression of dominance as influenced by soil fertility 133 over Imperata as compared to soil where Imperata dominates. This suggests that improvement in the soil is needed for Saccharum to dominate Imperata and that pure stand of Saccharum can develop in productive sites after eradicating Imperata through competition. The same was observed by Quimio (1996) but all these were based on field survey. It is not yet tested by experiment whether improvement in soil productivity in mixed Imperata-Saccharum stand would actually tip vegetative dominance in favor of Saccharum. This study was, therefore, aimed to determine whether improvement in soil through fertilization would favor Saccharum into gaining competitive dominance against Imperata.

Review of Literature

Imperata cylindrica and Saccharum spontaneum as plants

Little is known about the specific effects of light duration and intensity on the morphology and physiology of Imperata (Holm et al. 1977). But, it is generally recognized to be a light-loving plant which can be shaded out by a heavy canopy. The rhizome of Imperata contributes to its persistence once established, thereby making it hard to remove or eradicate. During grassfire, the rhizome is protected by soil against damage. Cutting of rhizome into pieces and loosening of the soil during plowing can even promote multiplication into separate individuals (Quimio 1996).

In relatively good sites, Imperata can produce thick canopy of leaves, thus also producing high amount of fuel for more intense burning. Instead of starting from germination of seeds after burning, it reclaims dominance on the land by sending up new leaf canopy through mobilization of energy stored in the rhizome. This gives Imperata the advantage in competition for light since it can consolidate its leaf canopy ahead of the other weeds. Fire can also damage the soil seed bank of other species and this further reduces their chance to compete with Imperata. Possession of underground node buds in the stubbles made Saccharum adapted to frequent fire damage and capable to stay and compete with Imperata in cogonal lands.

In productive sites, Imperata can develop an almost pure stand or stand that is low in number of species. In relatively poor to poor sites, Imperata can fail to express canopy dominance and so it grows together with high number of species.

In poor sites, where plants could not effectively express dominance, the tendency is for high number of species to co-exist; while in productive sites, the tendency is for highly competitive species to dominate at the expense of removing the others through competition (Grime 1987). There is also competition in co-existing 134 Quimio et al. plants that concentrates on soil resources. But in productive sites, competition shifts into above-ground resources, particularly for sunlight and space.

In ecological succession, earlier species modify the environment resulting in conditions disadvantageous to existing ones but favorable for establishment of another set of species (Odum 1971). The flux of nutrients from the ash after fire provides seasonal high availability of mineral elements in regularly burned areas (Sanchez 1976). Aside from better soil nutrient status, much improved soil structure is reported by Jayme (1998) in plots where Saccharum has expressed canopy dominance compared to plots where Imperata and Saccharum grow together. This is consistent with the consequences of succession wherein the improvement in soil conditions during the Imperata stage can favor the invasion and establishment of Saccharum. The succession model developed by Quimio (1996) indicated that occurrence of Imperata precedes Saccharum. The model hinges on the assumption that there is improvement in soil chemical and physical properties in Imperata- dominated grassland while under fallow. This is favorable towards colonization by Saccharum, and then eventually, removal of Imperata as Saccharum exerts greater dominance with further improvement in soil productivity. Quimio’s model was based mainly on field survey. It can be considered theoretical without confirmation by experimentation and test on whether Saccharum can really reduce Imperata as soil improves in productivity is necessary (Jayme 1998).

Site-indicating plant communities

Quimio (1998) has identified the ecological species and species groups that are associated to a combination of site factors, such as elevation, soil moisture regime, soil type, soil pH, soil geologic parent material, vegetation type, and management disturbance regime. An ecological species group is composed of species with more or less similar site range of occurrence, such that when one is absent on a site, the others are also absent. This made species groups to be associated to certain site factor range, and, thus, good site indicators. Quimio’s report was based on 432 plots, taken in 16 sampling sites that included five islands in the Visayas and three provinces in Luzon. Quimio’s (1996) model portrayed the sequence of succession among species groups as influenced by soil moisture regime, disturbance history, and age of vegetation. His data also indicated the need to correct the common thinking that Imperata grasslands are always in acidic soils. Imperata grassland is also widespread in basic limestone soils.

A study conducted by Quimio covered 16 sampling sites in Luzon, Visayas, and Mindanao. Plant communities are determined and named after their associated ecological species groups. Since species groups are site indicators, plant communities are also useful site indicators. Understanding the site-indicating value of Expression of dominance as influenced by soil fertility 135 plant communities in grasslands can reduce the need for costly and time-consuming laboratory analysis, such as for soil chemical and physical characterization. Plant communities can also be mapped in the field and such map could then be the basis in planting designs (Quimio 1996). Site-species matching could also be made easier to apply by simply matching species to plant with plant communities on site. Variation in productivity potential over a parcel of land can therefore be easily detected just by visual inspection of occurring plant communities. In contrast, if any such variation needs to be determined through laboratory tests, much more intensive sampling would be needed. That would mean more investments and waiting time before planting decisions can be finalized.

The current lack of understanding by field technicians on the applicability of grassland plant communities and vegetative formations to site-species matching could have forced them to ignore variability in site potentials even in highly variable, irregular, sloping lands (Quimio 1996). Thus, many reforestation sites, regardless of the different growth conditions, are being planted with the same species. However, in the ongoing National Greening Program that encourages planting of high number of native tree species, the need for more realistic ways on how to prepare field plans that considers site-species matching for a highly diverse planting design becomes more apparent. The use of Imperata cylindrica as site indicator of denuded grasslands makes no sense in site-species matching. Imperata cylindrica is widespread all over denuded lands; but within denuded lands, it loses the power of differentiating variation in site potentials. Imperata occurs in dry, moist, and wet sites, in low to high elevation, in poor to relatively good soils, shallow to deep, and acidic to basic soils. This made other species that grow together with Imperata but showing more confined or narrow to moderate range of occurrence to be better site indicators and useful in detecting variation in growth potential among sites. These species are those referred above as the ecological species groups.

Grassland research

Four community types in Philippine grassland were reported by Sajise in 1972. The species composition of such community types was, however, not presented. Two of the community types, the Themeda triandra and Capilepedium parviflorum community types were, however, found by Quimio (in press) as not typical to native grasslands by being more or less confined only in limited parts of Northern Luzon. Despite high interest in the development of Philippine grasslands in the 1980s to 1990s, the exhaustive literature review undertaken on the subject matter by Quimio (1996) discovered the very little published works on vegetation dynamics in local grasslands. The earlier works of Sajise and his students (Sajise 1972; Sajise, Palis, and Lales 1974; Sajise et al. 1974; Sajise and Lales 1975; Sajise et al. 1976 and Sajise 1980) were more related to agriculture and forage crops and less to grassland dynamics. 136 Quimio et al.

Beside the works of Quimio and his students on vegetation description and analysis, no other paper on species composition and site description in native grasslands can be found in local literature. This could be due to the presumption that our grasslands are already much described and understood, which is not correct. The presumption that site potential within grasslands is uniform has no basis, and, therefore, planting grassland areas with same exotic tree species also lacks ecological basis. There is always a concern, that when the subject of a study is on vegetation, the description on growth environment on site is not given due attention. Similarly, those studying soils in grassland often miss to give even a general description of natural vegetation on site. This deprived the possibility of generating data that would allow determination of association between site factors and species, again with the view of identifying site indicator species for the local grasslands.

Much works about native grassland were published through the Journal of the Grassland Society of the Philippines. The society was based in the Ecosystems Research and Development Bureau but was able to publish only in 1997 to 2000. The common topics were on soil erosion control, forage production, rangeland crops, plantation establishment, community-based grassland rehabilitation strategies, and agroforestry systems. But again, only Quimio and his associates (Quimio 1998; Quimio et al. 1998a; Quimio et al. 1998b; Quimio et al. 1998c; Quimio 2000) touched on vegetation description and grassland dynamics. The survey of articles in Sylvatrop Journal series also indicated the low interest in research on grassland dynamics. The interests as may be seen on topics of research outputs could be attributed to the trends in the direction of government programs in the past. In the 1980s to 2000s, the forestry development programs were focused on community- based upland rehabilitation. Unfortunately, the program implementation was biased towards empowerment in the social component while the technical side of studying vegetation dynamics in grassland was given comparatively less attention. By the turn of the century, the interest shifted from the use of exotic trees in plantations into high-diversity planting with native trees and carbon sequestration as it is now in the National Greening Program of President Benigno Aquino III. Therefore, it is hard to expect that interest in research on dynamics of native grassland would prevail this time. Nevertheless, one basic requirement to the development of a resource is the thorough knowledge on the material one is working on. Expression of dominance as influenced by soil fertility 137

Methodology

Experiment site

The study was conducted in a grassland area at the forest reservation of Visayas State University, Baybay City, Leyte. The site has Type IV climate, with average annual precipitation of 2,500 mm that is more or less uniformly distributed throughout the year (Fig. 1). The site has elevation of 60 m and slope of 10º. The soil is very deep, well-drained, clay loam, slightly acidic, and derived from basalt. Soil moisture is considered not a stress factor to limit growth of plants in the area.

The site was selected for the experiment based on five main considerations: 1) adequately large area that has vegetation showing fairly homogenous spatial distribution of Imperata and Saccharum; 2) dominance by Imperata and containing sparse but adequate representative bunches of Saccharum; 3) more or less uniform spread of Saccharum to allow adequate representation in all plots; 4) adequate distance from tall trees to avoid shading effects; and 5) relatively uniform character of the land.

Experiment design

The experiment had six 4 m x 4 m plots arranged randomly, three for control plots and three for treatment plots. Drainage canal, 20 cm wide and 15 cm deep, was dug around the plots to prevent contamination of control plots by applied fertilizer in treatment plots. At the center of each plot, the inner 2 m x 2 m area was marked at four corners by steel rods and the perimeter was delineated using blue nylon string. These marked inner 4 sq m in the plots were the measurement plots in which data collection was undertaken during the harvesting of Imperata and Saccharum. This made measurement plots to be 2 m away, edge to edge to reduce boundary effects. A composite soil sample was taken before treatment application to determine soil pH, percent organic matter, percent total nitrogen, and available phosphorus. Soil sample was also taken from each plot at the end of the experiment for the same soil analysis.

All plots were cleared by cutting all aboveground parts of Imperata and Saccharum at soil surface level. No data on biomass was taken during the clearing of the plots in July 1998. Plants were then allowed to grow without treatment application for five months, or until December 1998. Data collection and harvesting of biomass were undertaken this time. The first harvest intends to establish whetherImperata and Saccharum in control plots and in treatment plots were not statistically different in terms of the parameters used in data collection before the application of treatments. Data collected in the first harvest of Imperata and Saccharum were percent plot 138 Quimio et al.

Figure 1. Location of the sampling site and the climate map of the Philippines. Expression of dominance as influenced by soil fertility 139 cover, height of leaf canopy, number of shoots, and oven dry weight of biomass. The harvesting of biomass was done on per height strata basis, such that biomass was harvested separately at above 2 m, 1.5 m to 2 m, 1 m to 1.5 m, and below 1 m. The harvested biomass was then segregated according to species, oven-dried and weighed. The same data collection procedure was applied in the second and third harvests that followed.

After the first harvest, plants in plots were allowed to grow for five months again, or until May 1999, but now with application of fertilization treatments. One kg 14-14-14 NPK fertilizer was applied over the 4 m x 4 m area assigned for each of the treatment plots. The intention in this second harvest or data collection was to determine the effect of fertilization to growth performance of Imperata and Saccharum in short duration, or when Saccharum has shorter time to impose its competitive cover dominance potential against Imperata.

For the third harvest, treatment plots were again applied with 1 kg 14-14- 14 NPK fertilizer and the plants were allowed to grow for one year or from May 1999 to April 2000. This time, the intention was to determine whether Saccharum can impose competitive dominance over Imperata when allowed to grow without disturbance within the time interval commensurate to the regular burning cycle in grassland, which is every summer of each year. While it would be ideal to continue the experiment for another year, it was unfortunate that the land was already needed by the owner for other use.

The t-test was used to determine statistically significant difference between treatment means in each of the harvesting periods. Analysis for statistical difference of means between harvests was not undertaken. The reason was that the first harvest had no fertilization in all plots while the second had the treatment. Though both had same treatments, the second and third harvests differed in duration of growth before time of harvest.

Results and discussion

Table 1 shows the plot means on percent plot canopy cover, canopy height, number of shoots, and aboveground biomass of Imperata and Saccharum in the three harvesting events. The data in first harvest showed that plots selected for control and those for fertilization treatment were not statistically different in terms of the four parameters used for measurement of Imperata and Saccharum. This has established the assumption of similarity between control plots and treatment plots before the implementation of experiment treatments, a requirement to attribute difference in plant responses as resulting from effect of applied treatments in the second and third 140 Quimio et al. harvests.

Table 1. The percent plot canopy cover, canopy height, number of shoots, and oven dry biomass of Imperata cylindrica and Saccharum spontaneum in three harvests. Parameter Harvest period Plot canopy cover (%) Canopy height (m) Control Fertilized Control Fertilized a. Imperata First 92.0 88.0 1.43 1.33 Second 93.0 100.0 1.55 1.74 Third 35.0 25.3 1.50 2.00 b. Saccharum First 10.0 10.0 1.88 2.05 Second 5.0 23.0 2.12 2.22 Third 50.0 46.7 2.40 2.60

No. of shoots Dry biomass, t/ha Control Fertilized Control Fertilized a. Imperata First 297 298 10.10 9.83 Second 284 476 11.77 17.43 Third 521 304 5.15 3.97 b. Saccharum First 42 67 2.13 1.77 Second 31 97* 1.87 7.60* Third 83 139* 7.97 13.47*

Data in second harvest (Table 2) showed higher growth of Imperata when compared to averages in the first harvest, both in control and fertilized plots, in terms of plot canopy cover, canopy height and biomass production. Imperata had more shoots in the treatment plots but less shoots in control during the second harvest than in the first harvest. Less shoots of Imperata at control plots of second harvest, however, were compensated by having longer and broader leaves. Better growth of Imperata in second harvest, for both control and treatment plots, can be attributed to good weather conditions following the first harvest, which fell in December 1998. Expression of dominance as influenced by soil fertility 141

Precipitation at the site was highest in December and January. Since growth of Imperata has improved both in control and fertilized plots, no statistical difference between means on Imperata was detected in the second harvest.

While Imperata had shown better growth in control and fertilized plots at second harvest, Saccharum had less shoots, lower percent plot cover and lower biomass production in the control plots. These lower values on the growth of Saccharum in second harvest can be partly attributed to physiological effects of removal of aboveground parts during the first harvest, particularly in unfertilized plots. At harvest, the whole cane of Saccharum is cut and it takes time to produce new shoots from buds of the stubble. Unlike in Imperata, mostly the leaves were cut instead and new canopy of leaves are back in just a few days. On the treatment plots, however, Saccharum had higher growth at second harvest than the first harvest. Saccharum had significantly higher number of shoots (97/sq m) and biomass production (7.60 t/ ha) in treatment plots as compared to the control plots (Table 1). The control plots had means of 31 shoots/sq m and 1.87 t/ha oven dry biomass. Statistical significant response to fertilization was detected for Saccharum in the second harvest, but this was not so for Imperata. In the second harvest, Imperata had higher number of shoots and biomass production in the fertilized plot than the control plots but the difference was not statistically significant. Unlike in the third harvest,Saccharum was still building up its spread in terms of new shoot formation and has not yet imposing competitive dominance over the Imperata at the time of the second harvest.

In the third harvest, where plants in plots were allowed to grow for a year, Imperata had reduced growth while Saccharum showed an increase in all four growth parameters used. Saccharum had significantly higher number of shoots (139/sq m) and biomass production (13.47 t/ha) in the treatment plots. Control plots had means of 83 shoots/sq m and 7.97 t/ha dry biomass. This figure on biomass production of Saccharum is still much less compared to 34.22 t/ha reported by Napiza (2000) for a pure stand of Saccharum, also in Leyte. Due to longer time before harvest, Saccharum had more chance to attain its maximum growth potential and had longer time to impose vegetative competitive advantage against Imperata. This resulted in reduced growth of Imperata in fertilized plots. While Imperata had higher number of shoots in the control plots and comparatively similar leaf canopy height in third and second harvest, Imperata had less leaves per shoot, thinner leaves, lower percent plot canopy cover, and reduced biomass production in the third harvest.

Table 2 shows the biomass distribution of Imperata and Saccharum in four height strata. The leaf canopy of Imperata was concentrated at below 1.5 m height while the leaf spread of Saccharum was at 2 m and over especially in the fertilized plots at third harvest. The total biomass of Imperata and Saccharum in the third harvest was lower than in the second harvest. This can be partly due to losses in the 142 Quimio et al. form of decayed litter from over leaves of Imperata and Saccharum and dead canes of Saccharum. This did not happen in the earlier two harvests because of shorter growth period before harvest. The wet climate on site makes litter decomposition very fast.

Table 2. Plot means on harvest from standing biomass (tons/ha) of Imperata cylindrica and Saccharum spontaneum, by height strata. Height strata 1st harvest 2nd harvest 3rd harvest A. Control plots Imperata >1.5 m - 0.03 - 1 – 1.5 m - 1.27 0.06 < 1 m 10.10 10.47 5.07 Subtotal 10.10 11.77 5.13 Saccharum > 2 m - - 0.40 1.5 - 2 m 0.10 0.10 0.70 1 – 1.5 m 0.40 0.40 1.57 <1 m 1.63 1.37 5.30 Subtotal 2.13 7.97 B. Treatment plots Imperata >1.5 m - 0.30 0.20 1 – 1.5 m - 2.83 0.30 <1 m 9.83 14.30 3.47 Subtotal 9.83 17.43 3.97 Saccharum > 2 m - 0.06 0.57 1.5 - 2 m 0.13 0.37 0.97 1 – 1.5 m 0.41 1.80 1.87 <1 m 1.23 5.37 10.06 Subtotal 1.77 7.60 13.47 Total 23.83 38.70 30.53 Expression of dominance as influenced by soil fertility 143

No conclusive change in soil chemical properties before and after the experiment (Table 3) was detected in this study, though there is an indication of increase in soil organic matter. A total of 21 other weed species had been listed to have occurred in the plots during the experiment. Their combined contribution to biomass production in plots was considered insignificant, commonly less than one percent of the total. Most were ephemeral plants that were also subdued through competition by Imperata and Saccharum. Four climbing species were able to tolerate dominance effects of Imperata and Saccharum. These were Lygodium japonicum, Mikania cordata, Merremia tridentata, and Hewittia sublobata.

Table 3. Chemical analysis of soil before and after the experiment. pH OM (%) Total N (%) P (ppm) a. Before treatment, all plots 5.15 6.00 0.22 1.89 in composite sample b. At the end of the experiment Control plot 5.17 9.22 0.27 1.27 Treatment plot 5.08 8.80 0.24 1.75

The importance of plant height, potential maximum size, lateral spread and strategies to win the race to the canopy for access to above-ground resources as presented by Grime (1987) was very meaningful in this study. He also cited the tendency of associated plants to co-exist in relatively poor sites and the reduction in diversity due to expression of dominance by highly competitive species in good sites. In poor soils of Philippine grasslands, Saccharum is observed to be incapable of effectively imposing competitive dominance to the exclusion of Imperata and, hence, stays in mixture with Imperata (Quimio 1996). Along Imperata-Saccharum vegetation gradient, Jayme (1998) reported the trend of having more of Saccharum and less of Imperata at the more productive part of the gradient. Quimio (1996) also reported that pure stands of Saccharum normally occur in well drained soil, soil with highly developed soil crumb layer, and in dark colored soils that are indicative of organic matter accumulation over many years of regular burning.

Results of this study provided a strong support to the grassland succession model developed by Quimio (1996), which suggests Imperata formation would be replaced by Saccharum formation as soil nutrient and structure improve over time under the fire-regulated fallow system in Philippine grasslands. Once established, Saccharum would maintain dominance for as long as regular burning, its associated disturbance factor, is also maintained. Possible ways on how to deal with Saccharum in reforestation site are presented in the recommendation part of this paper. 144 Quimio et al.

Conclusion

This study concludes that improvement in soil productivity can tip the balance of competitive dominance in Imperata-Saccharum vegetation formation in favor of Saccharum and at the expense of reduction in growth or eventual complete removal of Imperata through competition. The result of the study supports the grassland succession models that portray succession of Saccharum in Imperata grassland by improving soil productivity after many fallow years under fire disturbance regime.

This is consistent with the observations in field survey research that areas dominated by Saccharum commonly have deep soils, well-drained, well-developed crumb structure, and are dark in color due to accumulation of organic matter through the years of regular burning. Furthermore, the results of the study support the grassland succession model of Quimio (1998), which reported that only Saccharum spontaneum can invade to the total exclusion of Imperata when regular burning is maintained. Woody plants cannot survive intense fire that is supported by dense biomass fuel of Imperata-Saccharum mixture and reestablishment through seeds is always prevented by fire. Saccharum has much bigger potential full size at maturity than Imperata and while Imperata has underground rhizomes, Saccharum has underground buds in its stubbles from which it grows again every after fire incident.

In poor soils, Saccharum could not express canopy dominance over Imperata and the vegetation would maintain Imperata-Saccharum mixture. Since light can still reach the ground, annual herbs may still persist as part of the species composition of the vegetation. In good soils or in poor soils that have undergone improvement in productivity through the years of being under fallow, it can be expected that Saccharum can completely over-shade and eventually totally exclude Imperata through competition. This is consistent with Grime (1979), such that when ground resources become less limiting, competition for above-ground resources, such as space and light, becomes more important.

Recommendations

Survey type of research on grassland vegetation should be encouraged. Results of the survey may serve as basis in generating hypothesis for controlled experiments. It is also recommended that researchers conducting studies on vegetation, be it in grassland, reforestation areas, or in forests, should provide a complete listing of species identified; and the growth factor situation on site should also be properly described. This practice should also be applied when reporting the growth performance of planted trees. When growth factors in sites are adequately described in field reports, together with survival and growth performance of the planted tree species, site range Expression of dominance as influenced by soil fertility 145 at which certain tree species could be expected to perform well when planted can be determined. If this will be a common practice among field workers in grassland, solid database for site-species matching can be generated and the hit-or-miss situation in assigning species to sites could be avoided.

Basic research on grassland succession needs more serious attention to have better understanding and basis for more effective rehabilitation strategies in Philippine grasslands, particularly in view of site-species matching. To do this, researchers would need the capability not only in measuring the parameters of vegetation data but also the field methods in the characterization of sites and plots. To compare results of field researchers working in varied growth environments, there must be a unified and agreed common methodology. Conduct of trainings on methods for studies on grassland dynamics is also highly recommended.

Meanwhile, with the understanding that soil in Saccharum-dominated areas has improved productivity potential than those in pure Imperata grasslands, it can be wise to plant fast-growing tree species that produce thick leaf canopy and are fire tolerant in Saccharum grasslands. Unfortunately, these characteristics are often found only in introduced exotic species, such as the yemane (Gmelina arborea) and mahogany (Swietenia macrophylla). The exotic Acacia species can produce thick canopy but it has thin bark to withstand burning. Most native species, even the pioneer trees, have thin bark that are sensitive even to low flame fires. Fire preventive measures should be maintained in young plantation until such time that the grass is subdued by overshading. In this manner, intensity of fire, if it occurs, would be greatly reduced and less damaging to growing trees. The strategy of relay reforestation may be applied, such that the fast-growing exotics would be planted first to out-shade the grass and then this can be followed by under-planting with native tree species. If diverse planting is the ultimate goal in forest land development, it would be too risky to plant native tree species directly in highly flammable Saccharum grassland even with highly elaborate and expensive fire prevention measures.

Trees with the characteristics of giant ipil-ipil (Leucaena leucocephala) can also be planted in Saccharum-dominated grasslands. Ipil-ipil can regrow from stump and other parts that may survive damage from fire. It produces profuse rain of seeds, which can remain viable in the soil seedbank. Ipil-ipil can produce seeds within a year from planting and start establishing its seedbank and a carpet of developing seedlings. Many pioneer tree species can also produce seeds early and capable of sprouting from stubble after fire. However, the staggered maturation of seeds presents a problem in seed collection.

Saccharum can be battered down by its associated vines species, particularly Mikania cordata and Calopogonium mucunoides. When Saccharum stand is relieved 146 Quimio et al. from fire, these vines can overwhelm the grass canopy and make the subsequent fire low in quality. As the weight of the vines pushes down the grass leaf canopy, sunlight can reach the ground and this can trigger germination of pioneer species from the soil seedbank. Fire maintains the dominance of Saccharum and removal of the fire influence on site can eventually give ecological succession a headstart to proceed.

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1. Acidity, conductivity and ionic trends of rainwater in acid deposition monitoring sites in Los Baños, Laguna and Quezon City, Philippines by Engr. Arcely C. Viernes and Engr. Maricris T. Laciste Reviewers: Ms. Lerma L. Dimayuga (EMB) Ms. Perseveranda-Fe J. Otico (EMB)

2. Trace metal speciation by sequential extraction in marine sediments of Calancan Bay, Sta. Cruz, Marinduque, Philippines by Dr. Dahlia C. Apodaca et al. Reviewers: Ms. Remy R. Mamon (EMB) Ms. Perseveranda-Fe J. Otico (EMB)

3. Growth performance of three eucalyptus (Eucalyptus deglupta x E. pellita) hybrids on half-sib progeny trial in Northern Mindanao, Philippines by For. Albert A. Piñon et al. Reviewers: Prof. Pastor L. Malabrigo, Jr. (UPLB-CFNR) For. Maura D. Dimayuga (ERDB)

4. Assessment of tree species diversity and structure of selected government reforestation projects by For. Manolito U. Sy et al. Reviewer: Dr. Manuel L. Castillo (UPLB-CFNR)

5. Genetic diversity of Limuran (Calamus ornatus Blume var. philippinensis Becc.) populations from three sites in Luzon Island, Philippines using Random Amplified Polymorphic DNA (RAPD) markers by Dr. Maria Theresa A. Delos Reyes et al. Reviewers: Dr. Ma. Genaleen Q. Diaz (UPLB-IBS) Ms. Faith S. Maranan (UPLB-IBS)

6. Expression of dominance as influenced by soil fertility in Imperata cylindrica-Saccharum spontaneum vegetation formation by Dr. Justino M. Quimio et al. Reviewer: Dr. Inocencio E. Buot, Jr. (UPLB-IBS) Sylvatrop Editorial Board

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