Freshwater microcrustaceans ( Cladocera : Anomopoda and Ctenopoda , Copepoda : Cyclopoida and Calanoida ) in the highly urbanized Metropolitan Manila area ( Luzon , Philippines )

Despite the increasing interest in studying Cladocera and Copepoda in Philippine freshwaters, there is a need to update our knowledge on its taxonomy and distribution in highly urbanized areas, such as Metropolitan Manila. This paper presents an updated listing of freshwater microcrustaceans and their distribution in Metropolitan Manila, considering the continued deterioration of water quality and increased urbanization in many areas since the last comprehensive study on these taxa in 1950s. We collected water samples from 33 freshwater sites in Metropolitan Manila and 23 of which were found to contain microcrustacean zooplankton. A total of 13 species were identified including two new locality records for C. cornuta and D. dubium in Pasig River. All 8 cladocerans, we identified have already recorded in previous studies while 3 cyclopoid copepods are new records for Metropolitan Manila. Furthermore, 16% of all known freshwater microcrustacean zooplankton species in the Philippines are found in Metropolitan Manila, including the Luzon-endemic Filipinodiaptomus insulanus collected from man-made reservoirs in less-disturbed habitats (an urban wildlife park, golf course and eco-park). Other identified species such as Thermocyclops taihokuensis may serve as indicator for high levels of nutrient. These results point to the importance of monitoring urban aquatic biodiversity for their potential in determining aquatic ecosystem health and of maintaining urban aquatic sanctuaries which may serve as alternative habitat for flora and fauna in rapidly developing urban centers such as Metropolitan Manila.


Introduction
Metropolitan Manila, with a land area of 633 km² (Manasan and Mercado 1999), is the most populated region in the Philippines due to the active migration of people from rural areas seeking livelihood in the metropolitan area after the Second World War (Vallejo et al. 2009).Its human population increases 1.1% every year, with a population of nearly 22 million as of 2012 (Zoleta 2000, Vallar et al. 2015).The increase in population and rapid urbanization have raised several environmental concerns, including the deterioration of environmental quality, particularly in freshwater ecosystems (Qian et al. 2000).According to Orozco and Zafaralla (2012), most rivers and streams in Metropolitan Manila are polluted due to urban use, various human activities and high discharge of organic materials from industrial and residential areas.Due to the growing problem of degrading water resources in the Philippines, including the dense area of Metropolitan Manila, several programs and policies were mandated to monitor and provide clean water (Rola et al. 2015).The implementation of policies involves the protection of the water bodies from pollution and provide water quality management to avoid loss biodiversity in these types of ecosystems.
Cladocera and Copepoda are widely distributed planktonic microcrustaceans that predominantly occur in freshwater.These taxa include littoral, pelagic and benthic species, which perform key roles in the food web.They have great value for fisheries, and in maintaining ecosystem processes.They also can be used as environmental indicators of pollution (Parmar et al. 2016), as they readily respond to changes in water quality.The changes in population density, species richness and community structure of cladocerans and copepods are mostly affected by the physicochemical conditions of the water in which they live.The reproduction and survival rates of cladocerans and copepods have a seasonal pattern that are highly dependent on favorable environmental conditions.Due to various anthropogenic activities, upwellings and runoffs of nutrients from tributaries, the trophic state of freshwater habitats may be altered.As such, these environmental changes influence the timing, reproduction rate, and survival rate of these groups of aquatic animals (Varpe et al. 2007, Tordesillas et al. 2016).
Extensive research on the distribution of freshwater microcrustacean in the Philippines started as early as 19th century with the Wallacea-Tressler Expedition that focused on the Oriental cladoceran and copepod species in Southeast Asia (Wright 1928, Kiefer 1930, Brehm 1938, Kiefer 1938, Woltereck et al. 1941, Brehm 1942).The earliest record of a microcrustacean species in Metropolitan Manila was by Marsh (1932), who described an endemic cyclopoid copepod, Thermocyclops philippinensis.This was followed in 1954 by the first inventory of the freshwater cladocerans by Cheng and Clemente (1954).The records of Philippine microcrustacean zooplankton was updated through the work of Uéno (1966), who recorded 8 cladoceran and 2 copepod species from Lake Taal (Luzon Island), Petersen and Carlos (1984) with 15 cladoceran and 25 copepod species from major lakes in Luzon, and Lake Lanao in Mindanao.The most comprehensive works are Mamaril and Fernando (1978), and Mamaril (1986, 2001), who listed 125 freshwater microcrustacean species (49 cladocerans and 9 copepods) from the various bodies of freshwater in the archipelago.Despite the initiatives done by several researchers in the past to document for the diversity of microcrustaceans in the country, our knowledge of them is still rather limited.Thereafter, several studies on the local distributions of cladoceran and copepod species had been conducted to address these gaps and revised some taxonomic issues of misidentified species.Several researchers managed to collect samples from poorly unexplored areas for microcrustacean species in Luzon (Tuyor and Baay 2001, Mamaril 2001, Aquino 2008, Papa and Hołyńska 2013), major islands in Visayas (Dela Paz et al. 2016aPaz et al. , 2016b)), in selected areas in Mindanao (Tuyor and Baay 2001, Pascual 2014, Dela Paz 2016b).Collections were also conducted in groundwater dependent ecosystems in the country (Cavite et al. 2017, Lopez et al. 2017a, b).The most recent update on the taxonomic status and distribution of freshwater microcrustacean zooplankton revealed the presence of 81 species in the Philippines including 55 cladocerans and 36 copepods (Lopez et al. 2017a).However, we still lack an updated listing of freshwater microcrustaceans in Metropolitan Manila.As such, information on the species composition of microcrustaceans (Cladocera and Copepoda) in Metropolitan Manila collected under the present environmental conditions in the area is extremely lacking.In this paper, we provide an updated species list and provide information on the distribution of freshwater microcrustaceans (Cladocera and Copepoda) in an urban area at Metropolitan Manila.

Methods
Study sites.Our samples were collected from June to July 2016 from 33 freshwater sites including ponds, rivers, small streams, and canals, and lagoons in Metropolitan Manila.Details on selected physico-chemical characteristics and location of the selected sampling sites are presented in Figure 1 and Table A1 in the Appendix.
The collection of cladoceran and copepod was done using a conical plankton net (70µm) which was towed vertically or horizontally, depending on the depth of the sampling site.The samples were then preserved with 70% ethanol, and then stored in labeled screw-capped bottles.The coordinates of the sample sites were obtained using a GPS receiver and mapped using QGIS software Version 2.18.11 using Luzon 1911 as the geodetic datum.
All voucher specimens were deposited at the University Santo Tomas, Zooplankton Reference Collection (UST-ZRC).

Results
Thirteen species of freshwater microcrustaceans belonging to 7 families were identified in this study.This includes 8 cladocerans from 5 families: Bosminidae Baird, 1845, Chydoridae Stebbing, 1902, Daphniidae Straus, 1820, Moinidae Goulden, 1968, and Sididae Baird, 1850.In addition, 3 species of cyclopoid copepods belonging to family Cyclopidae Rafinesque, 1815 and 2 species of calanoid copepods belonging to family Diaptomidae Baird, 1850.Complete list of all identified species and the sampling localities where they were collected is shown in Table 1

Short description.
The head is large and rectangular in shape, with no clear demarcation between the head and body along the dorsal surface.Head shield is present.The eye is small near the rostrum.The long rostrum is fused with the immobile antennules, with a slight concavity between the 2 parts.The antennules have a characteristic pointed projection along the midline.The postabdominal claw is almost straight with 6 spines and the distal part finely ciliated.Morphological characteristics concur with the descriptions and illustrations of Fernando (1992) and Pascual et al. (2014).
Ecological distribution.Bosmina fatalis is widely distributed in lakes, rivers, creeks, and reservoirs.This species is a primary grazer in the freshwater habitats and has a good defensive mechanism (akinesis) against its predator which allows them to live in the pelagic zone despite of its small body size (Rizo et al. 2017).A1.Thermocyclops decipiens (Kiefer, 1929)

Short description.
The body is hemispherical in shape, with no clear demarcation between the head and the body dorsally.The head shield is present.The rostrum is relatively longer than the antennule.A pair of 3-segmented second antennules with 3 projecting setae is found inferior to the antennule.The terminal claw is almost straight, with small spines.The postabdomen is short with pointed denticles on the postero-ventral margin.The morphological characteristics concur with the descriptions and illustrations of Fryer (1968), Petersen and Carlos (1984), Fernando (1992), Smirnov (1996), and Dumont and Negrea (2002).
Ecological distribution.Chydorus cf.sphaericus is present in lakes, reservoirs, and pools.This species is found in the littoral vegetation and pelagic area but can survive eutrophic conditions.

Short description.
A cervical sinus is evident between the head and the body.The head is small and round in shape, with a pointed rostrum and pigmented patch of ocellus.The head shield is present.The antennules are fused with the ventral portion of the head.Fine ciliation is present at the postabdominal claws.The morphological characteristics concur with the descriptions and illustrations provided by Petersen and Carlos (1984), Berner (1985), Fernando (1992) and Dumont and Negrea (2002).
Ecological distribution.Ceriodaphnia cornuta is present in lakes and a reservoir.This species is a primary grazer in the freshwater habitats and has a great competitive power against with larger grazers (

Short description.
The head is almost rectangular in shape, with a convex dorsal portion with eye near at the ventral margin.The carapace is oval shape.The posteroventral margin bears narrow free flap and ungrouped marginal spines.The postabdominal claw bears 3 spines, with short proximal spine and the long distal spine.
The morphological characteristics concur with the descriptions and illustrations provided by Mamaril Sr. and Fernando (1978), Fernando (1992), Korovchinsky (2002), Dumont and Negrea (2002) Short description.This species is relatively larger than the 2 Thermocyclops observed in this study.The body is generally large at the prosome and narrows to the urosome.The lateral arms of the seminal receptacle are long and broad and are curved posteriorly; the anterior margin has wave-like indentations at the proximal; Transverse duct-like structure forms a "V" shape.This species is highly recognized by the several hairs present at the entire median length of the caudal rami.The medial apical spine of the P4 endopodite is longer than the lateral, with spinules present at the entire anterior surface of 1 side and few spinules at the medial surface of the other side.All the morphological characters concur with the description of Hołyńska (2003) and Dela Paz et al. (2016a).
Ecological distribution.Mesocyclops aspericornis is found in wide variety of habitats including lakes, ponds, swamps, and rice fields (Lopez et al. 2017).This species has a great dispersal capability but adequate in competitive abilities.

Short description.
The body is generally elongated with a long antennule that generally reaches the tip of the urosome.Male specimen: antennule is geniculated.Right exopodite 2 of P5 leg is relatively large and bears a long, thin, and strongly curved terminal claw on the posteromedian area; and with a short lateral spine protruding near the middle of exopodite 2. Female specimen: antennule is straight.The pair of P5 is symmetrical.All the morphological characteristics concur with the descriptions and illustrations of Dussart and Defaye (2001) and Papa et al. (2012).
Ecological distribution.Arctodiaptomus dorsalis is found in lakes and rivers.This species is an invasive copepod hence that can tolerate eutrophic condition (Papa et al. 2012).

Short description.
The body is generally elongated with a long antennule that generally reaches the tip of the urosome.Male: the antennule is geniculated; the antepenultimate segment of the right antennule bears an antepenultimate process which contains 5-8 teeth on the outer margin.The right exopodite 2 of P5 is larger and longer than the left, and has a long, broad, and slightly curved terminal claw on the postero-median area; and with a short lateral spine protruding from the exopodite 2. Female: the antennule is straight.The pair of P5 leg is symmetrical.All the morphological characters concur with the species descriptions of Lai and Fernando (1980).

Discussion
Among the cladocerans, members of the family Sididae including Diaphanosoma dubium, D. excisum, D. sarsi, were the most frequently encountered species from the collected samples, followed by moinids and daphniids such as Moina micrura, Moinodaphnia macleayi, and Ceriodaphnia cornuta.In contrast, Bosmina fatalis and Chydorus cf.sphaericus were rarely encountered in the samples, occurring only at three localities with the latter found in only 1 locality (Table 1).Chydorus cf.sphaericus is a known cosmopolitan species (Maiphae et al. 2008), which is tolerant to variable physico-chemical water characters, thus making it good bio-indicator.It mostly occurs in the littoral area with macrophyte stands.As such, the rarity of this animal in our collection may also be due to the absence of macrophytes during the sampling and the lack of collections benthic habitats.The 8 species of Cladocera identified in our study were already known to be present in Metropolitan Manila from surveys conducted between the 1950s and 1970s (Cheng andClemente 1954, Mamaril 1978) from La Mesa reservoir, as well as the Pasig and Marikina rivers.However, there are new locality records for C. cornuta and D. dubium in Pasig River (Table 1).The species composition and distribution of these species agrees with the previous observation of Fernando (1980a, b) on the distributional patterns of Diaphanosoma in comparison with other cladoceran taxa as well as the absence of Daphnia in the tropics.So far, the genus Daphnia are restricted to temperate and subtropical regions.The old record of the Daphnia pulex in the Philippines (Wright 1928) is suspicious and need validation (Petersen andCarlos 1984, Mamaril 2001).
Our current efforts in sampling Cladocera from various localities throughout the Philippines has not yielded any Daphnia so far (Cheng and Clemente 1954, Mamaril 2001, Pascual et al. 2014, Dela Paz et al. 2016b).
We collected copepod samples from small (ponds and canals) to large water bodies including Pasig River and La Mesa reservoirs (Table A1, Appendix) during the wet season.We were able to identify Mesocyclops aspericornis, Thermocyclops decipiens and T. taihokuensis from various localities (Table 1) together with 2 species of calanoid copepods, Arctodiaptomus dorsalis and Filipinodiaptomus insulanus (Table 1).
Previously recorded copepods in Metropolitan Manila include A. dorsalis (Pasig River) (Papa et al. 2012), F. insulanus (La Mesa Reservoir) (Mamaril 1986) and T. philippinesis (Manila) (Marsh 1932).Arctodiaptomus dorsalis and F. insulanus were still collected from various sites in Manila.However, the endemic T. philippinensis has not been identified from any of our collected samples.As such, no new occurrence of T. philippinen-sis has been found since the study of Marsh (1932).All identified cyclopoid copepods in this study, including M. aspericornis, T. decipiens and T. taihokuensis are new locality records for 15 sites (Table 1).These species are commonly collected from different freshwater habitats in other parts of the country (Papa andHołyńska 2013, Dela Paz et al. 2016b).The distribution of the fugacious M. aspericornis in the Philippines was previously limited to 1 record each on the islands of Luzon (Tuyor and Baay 2001), 1 in Camiguin (Hołyńska 2000), and 1 in Cebu (Dela Paz et al. 2016a).Hence, the occurrence of M. aspericornis in Metropolitan Manila contributes to the better understanding of its species distribution in the Philippines by providing new insights on its possible pattern of dispersal in the country and the entire Southeast Asian region (Table 1).The Philippine record of T. decipiens ranges from northern to southern Philippines including major islands such as Luzon, Panay, Negros, Cebu, Bohol, and Mindanao (Papa andHołyńska 2013, Dela Paz et al. 2016a, b).This species was found in a wide range of freshwater habitats including lakes, fishponds, dams, and waterfalls from previous study done by several researchers (Dela Paz et al. 2016a, Lopez et al., 2017).
On the other hand, T. taihokuensis was previously recorded from the northern and southern parts of Luzon (Papa andHołyńska 2013, Dela Paz andPapa 2018), in eastern Visayas (Dela Paz et al. 2016a), and southern Mindanao (Dela Paz et al. 2016b).This cyclopoid is commonly observed in lakes and fishponds.Hence, we suspect that aquaculture and co-introduction of planktivorous fishes which harbor zooplankton are the possible causes of introduction of this species in Philippine freshwaters.The occurrence of T. taihokuensis in a highly urbanized area such as Metropolitan Manila where freshwaters have high nutrient levels seemed to support the idea that T. taihokuensis can disperse and maintain its population in a eutrophic environment.As such, this species can be used for biomonitoring.A few studies of the plateau lakes in China revealed that the great biomass of copepods (including T. taihokuensis which contributed more on the total biomass because of its relatively high density) is positively correlated with the high level of Chlorophyll a (Wang et al. 2007, Guo et al. 2009).As an omnivorous animal, T. taihokuensis consume blue-green algae which most other copepods cannot exploit; this helps maintain high populations in mesotrophic or eutrophic water conditions (Reid 1989, Chang andHanazato 2004).Moreover, high biomass of algal bloom-forming phytoplankton species would cause a decrease on the amount of dissolved oxygen which provides a potential refuge of T. taihokuensis against planktivorous fishes (Chang and Hanazato 2004).Also, it had been observed that T. taihokuensis was one of the most common components of the plankton community during summer and this animal was found in lesser numbers or totally absent during winter in subtropical freshwaters (Wang et al. 2007, Guo et al. 2009).In the Philippine, T. taihokuensis has been found in ponds and lakes on Leyte Island and South Cotabato during wet and dry seasons (Dela Paz et al. 2016a, b) which points to the fact that this species occurs throughout the year in the tropics.As such, T. taihokuensis may indeed be considered as a thermophilic cyclopoid (Wang et al. 2007) given its predominance in tropical freshwaters.
The invasive calanoid copepod A. dorsalis was originally recorded from Americas and subsequently introduced in the Philippines through the release of drinking water reserves of transcontinental ships, as well as through the aquarium trade (Papa et al. 2012).The first record of this species in the Philippines was in Laguna de Bay (Luzon Island) (Papa et al. 2012).Current efforts to determine the distribution of A. dorsalis in the Philippines revealed that this species occurs in several lakes on Luzon and Mindanao (Tuyor and Baay 2001, Papa et al. 2012, Rizo et al. 2015, Dela Paz et al. 2016b).Our results revealed that A. dorsalis is not widely distributed in Metropolitan Manila except for Pasig River, which is the sole outlet of Laguna de Bay.On the other hand, the endemic F. insulanus was first collected in Manila (exact location not mentioned in the paper) (Wright 1928) and has thereafter been recorded from Laguna de Bay and La Mesa reservoir (Petersen and Carlos 1984, Mamaril 1986, Mamaril 2001), Lake Paoay (Aquino et al. 2008, Papa et al. 2012), Pantabangan Dam reservoir (Lopez et al. 2017) and Lake Danao in Leyte (Tuyor and Baay 2001) has been collected in a golf course lagoon and again from the La Mesa reservoirs in Quezon City (Table 1).The record in Lake Danao is a possible misidentification, since as previous records from Brehm (1933) revealed the presence of Diaptomus vexillifer in the lake.This was further validated in more recent studies as reported by Lopez et al. (2017).Filipinodiaptomus insulanus seems to be present only in less-disturbed habitats with low fish densities.Our results also confirmed the northern distributional range of F. insulanus in the Philippines.
Information on the occurrences of the Cladocera and Copepoda on the freshwater habitats in Metropolitan Manila helps us to better understand the ability of these animals to tolerate and maintain a population in the wastewater and contaminated water in urban areas.In comparison, the cladocerans C. cornuta, D. sarsi, M. micrura and the copepod T. taihokuensis were similarly found in urban ponds and wetlands in Turkey and southern China (Liu et al. 2016, Adhikari et al. 2017).Such observations have confirmed that these microcrustaceans can be regarded as "urban zooplankton", which may be useful indicators for assessing water quality.
Despite the poor water quality in most areas in Metropolitan Manila, our results show that 13 species of freshwater microcrustaceans are present at our 23 sampling localities.The presence of these species in highly urbanized areas like Metropolitan Manila points out capacity of these species to thrive in eutrophic, highly polluted, and disturbed aquatic environments.Though this study does not put emphasis on the relationship between the distribution and abundance of the collected specimens and the prevailing environmental conditions in the different sampling sites, the results still serve as an important update on the species composition of microcrustaceans in the highly urbanized area of Metropolitan Manila, where little or no attention has been given to studying freshwater biodiversity and conservation.Interestingly, all the cladocerans and copepods identified from Metropolitan Manila were already observed from other freshwater ecosystems in the Philippines (Dela Paz et al. 2016b).However, our current result revealed distinct patterns in terms of microhabitat choice by these microcrustacean zooplankton species in an urban environment.Given the low number of studies conducted on urban biodiversity and ecology, and the limited taxa covered by these studies (Vallejo et al. 2009) in Metropolitan Manila, our results serve as an important contribution to our current knowledge of aquatic biodiversity in Metropolitan Manila, one of the most densely populated urban centers in the planet.

Figure 1 .
Figure 1.Map of the selected sampling localities in Metropolitan Manila.Number of sampling site in the Appendix, TableA1.

Table 1 .
List of observed Cladocera and Copepoda and distribution in Metropolitan Manila.
Dela Cruz, GAA Viernes, JF Wong, ESP Dela Paz and ML Lopez, June 2016 (UST-ZRC 0190, 1 spec.).The body is generally large at the prosome and narrows to the urosome.The arms of the seminal receptacle are wide and straight.The caudal rami are smooth and naked.The marginal extension at the P4 basipodite bears few spinules.The distal margin at P4 intercoxal sclerite is high and triangular in shape.Thermocyclops taihokuensis is found mostly in lakes, small pool, fish ponds, and rice fields.This species can survive and tolerate eutrophic condition.