LSM3254 Ecology of Aquatic Environments Practicals 2 and 3: Stream communities and biomonitoring 6, 27 September 2011

INTRODUCTION Following up on Practical 1, in which representative freshwater fauna were examined and identification keys were used, this practical looks at local freshwater stream communities. In addition to experiencing and observing local freshwater streams, some freshwater sampling techniques are introduced. OBJECTIVES To survey freshwater stream habitats found near the edges of core nature areas in Singapore To observe and sample freshwater stream invertebrates and fishes To process (e.g., label, organise, sort, and identify) samples of freshwater stream invertebrates To describe the invertebrate community structure of freshwater streams using indices of species diversity 5. To estimate the relative water quality in freshwater streams using biomonitoring indices based on the invertebrate community 6. To make quantitative and qualitative comparisons of the communities, environments, and water quality of freshwater streams BACKGROUND Communities are assemblages of species that occur and interact in particular habitats. Stream communities involve aquatic/semi-aquatic species in stream habitats. Like most habitats, various microhabitats can be discerned within streams, each with their respective microhabitat communities. Stream microhabitats include channel, riffle/hyporheic, pool, periphyton, and riparian floodplain zones (for practical reasons, however, we will investigate the overall stream community and not differentiate between microhabitats in this exercise). A community can be described in quantitative or qualitative terms. Quantitative community parameters include richness (the number of taxa, usually species, present); abundance (total number of organisms); evenness (the relative abundance of taxa present), and diversity, which takes into account both richness and abundance. Taxonomic diversity can be described quantitatively using various indices. One commonly used index is the Shannon Index (H):
s

1. 2. 3. 4.

H = - pi loge pi
i=1

pi = proportion of the ith species; loge pi = natural logarithm of pi; s = number of species in the community

Diversity can also be illustrated graphically using rank-abundance curves, which involve plotting the relative abundance of taxa against their abundance rank. A community can also be described qualitatively from taxonomic or community composition. This can be in the form of a taxa list (usually, but not always, at species level). A taxa list tells the reader what taxa are present in the community/habitat, and contains information not normally conveyed by quantitative indices, e.g., changes in the types of taxa present over time even as the number of taxa (richness) remains constant (turnover); presence of a particular species of high
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conservation priority; presence of non-native species; and presence of unusual nor unexpected speciespossibly indicating sampling errors. A taxa list does allow richness to be determined, but does not give any quantitative information about abundance, dominance, or rarity. Taxa lists are nevertheless important because the qualitative sampling methods and protocols used to generate them are often better at detecting rare or highly patchily distributed species that might otherwise not be captured by quantitative sampling. Rivers and streams can possess diverse and abundant communities of invertebrates, which are convenient to sample. Freshwater stream invertebrates range from taxa that are highly sensitive to environmental disturbance (e.g., pollution) to hardy taxa that are tolerant of pollution. Because of this, stream invertebrates are often used as bioindicators of water quality in stream biomonitoring programmes. To assess water quality using macroinvertebrates, biotic indices, based on the presence and abundance of various taxa scored for their tolerance or sensitivity to various water quality conditions, are calculated. One such biotic index is SingScore, developed for freshwater streams in Singapore (Blakely et al. 2010). In SingScore, macroinvertebrate families in running waters are given tolerance scores from 110, 1 being the most pollution-tolerant and 10 being the most pollution-sensitive (see Table 3). Sensitivity here may be to organic pollution, which in turn affects dissolved oxygen availability To calculate SingScore for a site, total up the tolerance scores of the taxa and divide that by the number of taxa to obtain an average tolerance score for the sample or site, and multiply the figure by 20 (Blakely et al. 2010), i.e. SingScore = (total tolerance score/no. of taxa scored) x 20 SingScore values can be used as proxies for the water quality of streams. The scores generally correspond with categories of stream water quality as follows (after Blakely et al 2010): 079: poor water quality 8099: fair water quality 100119: good water quality 120: excellent water quality Two streams will be visited for this practical, one at the Bukit Batok Nature Park and another at Venus Drive (Central Catchment Nature Reserve or CCNR). Bukit Batok Nature Park is a small fragment of forest/park land west of Upper Bukit Timah Road, while Venus Drive is located west of Upper Thompson Road, on the eastern edge of the CCNR, the largest unbroken patch of forested land in Singapore. The National Parks Board (NParks) is interested in finding out more about these streams, e.g., the nature of the aquatic communities present. The data collected from these practicals will be communicated to NParks and will contribute information towards management of these freshwater resources. PROGRAMME This exercise runs over two practicals. The first practical involves a field trip to a freshwater stream in central Singapore to sample stream invertebrates and fishes, and measure physical and chemical environmental parameters of the stream. The class will be divided into two to visit two different streams (Bukit Batok Nature Park and Venus Drive). The practical that follows is a laboratory session to sort the invertebrate samples, and begin processing and analysing the data obtained to compare various aspects of the two streams. A report of the findings written in the format of a scientific paper is to be submitted before the next practical.

I. Field trip to forest stream at Bukit Batok Nature Park/Venus Drive (26 pm, 8 Sep 2010) A. Materials Per group: Pencil and labels for samples Photograph label/scale bar Plastic containers (5001000 ml) for samples x 6 Pail/large carrier bag for sample containers x 1 95% alcohol x 1.5l Tray net (0.4 x 0.6 m) x 1 Surber sampler (0.33 x 0.33 m) x 1 Black trash bags Spherical densiometer x 2 First aid kit x 1 Insect repellant x 1 Digital/phone camera x 1 1 metre ruler x 1 Tape measure (3050m) x 1 Per site (shared among four groups at Bukit Batok or at Venus Drive): GPS unit x 1 Multimeter probe x 1 B. Procedure The class will carry out a survey of the stream invertebrate and fish communities and environmental parameters. As time is limited in this practical, each group will only survey one 20 m stretch or transect within the stream, with four quantitative Surber samples (two in middle of the stream, two at the stream edges) and four qualitative tray net samples (all four at the stream edges). For each transect, each group should do the following: 1. Plan before starting. Identify and discuss among yourselves and with your Teaching Assistant (TA) the location of the transect. Subdivide the transect into FOUR equal sections (A, B, C, and D). One set of data will be recorded in each section, i.e. one set of physical environmental parameters, one Surber sample, and one tray net sample (see Fig. 1). Identify and discuss the location of the sampling points for the Surber and tray net samples in each section (Note: two of the Surber samples should be in middle of the stream). Discussion question: How might location of the transect and samples influence the sampling results?
Section A = 5 m Section B = 5 m Section C = 5 m Section D = 5 m

Sample A

Sample B Direction of water flow Sample C

Sample D

Fig. 1. Example of sampling design in a 20 m stream stretch/transect by one group. Surber sampler Tray net 3

2. Before beginning sampling, record the location using a hand-held Global Positioning System (GPS) unit and the following chemical environmental (water quality) parameters using a multimeter from the midpoint of the transect and middle of the stream for the transect (your TA will guide you) in Table 1: 1) pH 2) Temperature (C) 3) Dissolved oxygen (mg/l) 4) Conductivity (microSeimens) Discussion question: How might such water quality parameters influence the macroinvertebrates and fishes obtained?
Table 1. Group data of stream location and chemical environmental parameters (one set per transect) Group/Transect no.: ____________ GPS coordinates pH Temperature (C) Dissolved oxygen (mg/l) Conductivity (microSeimens)

3. Make qualitative field notes and record the following parameters in each section of the transect in Table 2: 1) Bank vegetation (bare, grassy, riparian vegetation: herbaceous or woody?) 2) Canopy cover (%) using the spherical densiometer from the middle of the stream (your TA will guide you) 3) Estimated substrate composition (% gravel/cobble, sand, clay, mud, and/or concrete) 4) Estimated leaf litter (% cover) 5) Wetted width (m) 6) Wetted depth (m) 7) Surface flow rate (m/s) Discussion question: How might such physical parameters influence the water quality and the macroinvertebrates and fishes?
Table 2. Group data of stream physical environmental parameters (four sets per transect, one in each section) Environmental parameter Section A Section B Section C Section D Bank vegetation (bare, grassy, riparian vegetation herbaceous or woody trees) Canopy cover (%) Substrate composition (% gravel/cobble, sand, clay, mud, and/or concrete) Leaf litter (% cover) Wetted width (m) Wetted depth (m) Surface flow rate (m/s)

4. Collect FOUR quantitative samples using a Surber sampler, one from each section of the transecttwo in the middle of the stream and two from the stream edges (this is just to ensure that both these microhabitats are sampled in each transect, but we will not be comparing the two). These samples are to be brought back to the lab for sorting and identification. Working in teams of two: 1) Clean the Surber sampler before use, ensuring that no materials from previous samples are present.
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2) Starting from the downstream section, work your way upstream (co-ordinate with the tray net sampling team). Discussion question: What is the rationale for working in a downstream to upstream direction? 3) Place the quadrat frame of the Surber sampler on the bottom of the stream (making sure to minimise gaps between the frame and the bottom), with the net opening facing upstream (see schematic diagram in Fig. 2). 4) Sample for 1 minute by disturbing the substratum, i.e. sand, stones, macrophytes, leaf litter, etc., within the quadrat with a stick or your hands, to a depth of about 35 cm, such that macroinvertebrates and substrate flow downstream into the Surber sampler net. Larger items that may be in the quadrat (e.g., large stones, coarse woody debris) should not be pushed into the net, but instead their surface should be brushed/scrubbed in the flowing water to dislodge and wash attached macroinvertebrates into the net. 5) Deposit the entire contents of the Surber sampler net (although a little water may be necessary to wash the contents into the container, try to minimise the amount of water that is transferred into the container) into a properly labelled container that is half-filled with 95% ethanol (the ethanol can be topped up later if necessary). The label should include relevant collection information such as sample or section number, sampling method, location, microhabitat (middle or edge of stream), date, time and collectors (LSM3254 group number and TA name). Put the container into the pail or carrier bag. Note that each Surber sample from each section will be regarded as one replicate and must be kept in its own container separate from other samples. 5. Collect FOUR qualitative samples using a tray net, one from each section of the transect (all from the stream edges). Only invertebrates are to be brought back to the lab for sorting and identification. Working in teams of two: 1) Clean the tray net before use, ensuring that no materials from previous samples are present. 2) Starting from the downstream section, work your way upstream (co-ordinate with the Surber sampling team). 3) One student should place the tray net at the angle of the streambed and bank (making sure to minimise gaps between the frame and the streambed and bank), with the net opening facing upstream. Hold the tray net at a gentle diagonal angle, i.e. not perpendicular, to both the streambed and bank (outer and bottom edges further forward) (see schematic diagram in Fig. 2). 4) The other student should kick sample for 1 minute by kicking and disturbing the substratum and bank, i.e. sand, stones, macrophytes, leaf litter, submerged bank and bank vegetation, etc., from 2 m upstream of the net, working his/her way downstream towards the tray net, such that macroinvertebrates and fishes are disturbed into the tray net. The student holding the net must be alert and prepared to actively scoop up or raise the net if he/she notices large or fast-moving organisms entering the tray net. 5) Macroinvertebrates collected in the tray net should be picked out and transferred into a properly labelled container that is half-filled with 95% ethanol (the ethanol can be topped up later if necessary). Fishes and other vertebrates should be photographed in the net next to a label-scale bar for subsequent identification and must be released just downstream of the sampling site as soon as possible. The labels should include relevant collection information such as sample or section number, sampling method, location, date, time and collectors (LSM3254 group number and TA name). 6) Unlike the Surber samples, all the tray net samples will be combined into a single replicate for each group/transect, so macroinvertebrates from each tray net sample in each section can be put together in one container. Put the container into the pail or carrier bag.
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Surber sampler

Direction of water flow

Tray net Fig. 2. Schematic plan view of sampling methods

6. Ensure that all sample containers are properly labelled in the field. Upon returning to the laboratory, ensure that all your samples are properly organised and stored, and that your field equipment is washed and set out to dry. TAs will advise on this.

II. Laboratory Work in LS Lab 7 (26 pm, 27 Sept 2011) A. Materials Per group: White trays Forceps Disposable pipettes Petri dishes Vials Ziploc bags Labels Dissecting stereomicroscopes References for identification (identification guides and taxonomic keys) B. Procedure Surber samples The class will sort the Surber samples (four samples from each transect per group). For each sample, each group should do the following: 1. Pour the sample into a white tray of water, and separate macroinvertebrate specimens from substrate and debris by first removing plant fragments and debris from the tray (after checking for animals). Then start picking or sorting through the specimens. 2. Based on the identification keys in the guide to macroinvetebrates by Blakely et al. (2010), identify (to at least family level or where possible, to genus level) the macroinvertebrate specimens in your samples by doing the following: 1) Without using a microscope at first, carry out coarse sorting of the material into broad categories based on their overall appearance (morphology). 2) Then carry out a finer sorting under low power magnification using a dissecting stereomicroscope.
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3. Count the number of specimens in the taxonomic groups listed in Table 3, and enter the data into Table 3. Each practical group should have four sets of quantitative data from four samples/sections of the transect. Tray net samples The class will sort the tray net samples (one sample pooled from four sections from each transect per group). For each sample, each group should do the following: 1. Follow the same protocol as for Surber samples (see above). There is NO NEED to count the number of specimens. Just indicate presence with a tick in the last column in Table 3. Each group should have one set of qualitative data from the combined sample. 2. Identify fish photographs using the freshwater fish guidebook by Lim & Ng (1991) and enter the names into the empty rows at the bottom of Table 3.
Table 3. Group data of stream samples Taxon (with SingScore* tolerance value in parentheses) Platyhelminthes Polychaeta Oligochaeta Hirudinea Mollusca Dugesiidae (3) Nereididae (6) Tubificidae (2) Erpobdellidae (1) Glossiphoniidae (2) Haemadipsidae (5) Ampullaridae (3) Ancylidae (1) Bithyniidae (3) Lymnaeidae (1) Physidae (2) Planorbidae (2) Thairidae (1) Amphipoda (2) Atyidae (6) Copepoda (4) Isopoda (1) Ostracoda (3) Palaemonidae (7) Parathelphusidae (9) Amphipterygidae (10) Calopterygidae (8) Coenagrionidae (3) Euphaeidae (9) Platycnemididae (5) Platystictidae (8) Aeshnidae (9) Corduliidae (5) Gomphidae (8) Libellulidae (4) Baetidae (7) Caenidae (7) Heptageniidae (9) Leptophlebiidae (10) Leuctridae (10) Perlidae (9) Corixidae (10) 7 Surber sampler (enter no. of individuals) Sample A Sample B Sample C Sample D (Section A) (Section B) (Section C) (Section D) Tray net (tick to indicate presence only)

Crustacea

Odonata (Zygoptera)

Odonata (Anisoptera)

Ephemeroptera

Plecoptera Hemiptera

Megaloptera Trichoptera

Lepidoptera Coleoptera

Diptera

Collembola Acari

Gerridae (5) Helotrephidae (4) Mesoveliidae (10) Naucoridae (7) Notonectidae (8) Veliidae (7) Sialidae (8) Calamoceratidae (9) Ecnomidae (6) Hydropsychidae (7) Hydroptilidae (6) Leptoceridae (7) Odontoceridae (10) Philopotamidae (8) Polycentropodidae (9) Crambidae (5) Curculionidae (6) Dytiscidae (5) Hydraenidae (8) Hydrophilidae (6) Scirtidae (8) Athericidae (10) Canacidae (6) Ceratopogonidae (3) Chironomidae (2) Corethrellidae (10) Culicidae (1) Dolichopodidae (1) Empididae (4) Ephydridae (4) Muscidae (6) Psychodidae (2) Simuliidae (7) Syrphidae (4) Tipulidae (3) Collembola (5) Acari (4)

Total *After Blakely et al. (2010)

Pooling class data Each group will contribute to the overall data of either of the streams (Tables 4A, 4B) for the whole class to use.
Method Group Sample Table 4A. Class dataset (Groups 13) of stream samples from Bukit Batok Nature Park Surber sampler 1 2 3 4 Overall 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 n A B C D A B C D A B C D A B C D Mean SD SE Tray net 2 3 2 () 3 ()

1 1 ()

4 4 ()

Dugesiidae Nereididae Tubificidae Erpobdellidae Glossiphoniidae 8

Haemadipsidae Ampullaridae Ancylidae Bithyniidae Lymnaeidae Physidae Planorbidae Thairidae Amphipoda Atyidae Copepoda Isopoda Ostracoda Palaemonidae Parathelphusidae Amphipterygidae Calopterygidae Coenagrionidae Euphaeidae Platycnemididae Platystictidae Aeshnidae Corduliidae Gomphidae Libellulidae Baetidae Caenidae Heptageniidae Leptophlebiidae Leuctridae Perlidae Corixidae Gerridae Helotrephidae Mesoveliidae Naucoridae Notonectidae Veliidae Sialidae Calamoceratidae Ecnomidae Hydropsychidae Hydroptilidae Leptoceridae Odontoceridae Philopotamidae Polycentropodidae Crambidae Curculionidae Dytiscidae Hydraenidae Hydrophilidae Scirtidae Athericidae Canacidae Ceratopogonidae Chironomidae Corethrellidae Culicidae 9

Dolichopodidae Empididae Ephydridae Muscidae Psychodidae Simuliidae Syrphidae Tipulidae Collembola Acari

n = total no. of individuals; Mean = average no. of individuals/0.1m2 ; SD = standard deviation; SE = standard error Table 4B. Class dataset (Groups 46) of stream samples from Venus Drive Surber sampler 1 2 3 4 Overall 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 n A B C D A B C D A B C D A B C D Mean SD SE

Method Group Sample

1 1 ()

Tray net 2 3 2 () 3 ()

4 4 ()

Dugesiidae Nereididae Tubificidae Erpobdellidae Glossiphoniidae Haemadipsidae Ampullaridae Ancylidae Bithyniidae Lymnaeidae Physidae Planorbidae Thairidae Amphipoda Atyidae Copepoda Isopoda Ostracoda Palaemonidae Parathelphusidae Amphipterygidae Calopterygidae Coenagrionidae Euphaeidae Platycnemididae Platystictidae Aeshnidae Corduliidae Gomphidae Libellulidae Baetidae Caenidae Heptageniidae Leptophlebiidae Leuctridae Perlidae Corixidae Gerridae 10

Helotrephidae Mesoveliidae Naucoridae Notonectidae Veliidae Sialidae Calamoceratidae Ecnomidae Hydropsychidae Hydroptilidae Leptoceridae Odontoceridae Philopotamidae Polycentropodidae Crambidae Curculionidae Dytiscidae Hydraenidae Hydrophilidae Scirtidae Athericidae Canacidae Ceratopogonidae Chironomidae Corethrellidae Culicidae Dolichopodidae Empididae Ephydridae Muscidae Psychodidae Simuliidae Syrphidae Tipulidae Collembola Acari

n = total no. of individuals; Mean = average no. of individuals/0.1m2; SD = standard deviation; SE = standard error

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PRACTICAL REPORT Write a report for Practicals 2 and 3 (max. 5 pages excluding title page, figures and tables, and references) using the pooled class data collected at Bukit Batok and Venus Drive to calculate indices to describe the community structure of each stream and to make a comparison of the two streams. The format of the report (including how to cite and list references) must follow closely to that of the journal Aquatic Biology (See Author Instructions at http://www.intres.com/journals/ab/guidelines-for-ab-authors/ as well as refer to recent papers from the journal as a guide).This is not just a lab report; it should have the feel of a scientific paper. Your report must include the following sections: 1) Title page, including: a. Title of report (up to you to think of an appropriate title) b. Group number and Teaching Assistants name c. Matriculation number 2) Introduction, including: a. Brief background that i) includes a brief literature review and ii) presents the rationale for why and how you came up with your particular research questions and hypotheses. b. A short paragraph at the end, which clearly states your i) specific research questions (and remember to phrase these literally as questions); and ii) hypotheses (i.e. your predicted answers to your research questions). Discuss the possible questions and hypotheses with your TA. 3) Methods. This section should be written in the past tense and should describe how the study was done, including: a. Site selection and very general description b. Sampling methods c. Statistical tests used for data analysis Note: Do not include equipment lists. Do not copy from this practical schedule. To learn how to write a good Methods section (or for that matter, all sections of the report), read published papers of similar studies. 4) Results. Concisely present/compare the pooled class results (will be uploaded onto IVLE) of the two localities in words in conjunction with appropriate visual aids, e.g., tables, histograms, or the appropriate statistical test. These should include: a. Qualitative (descriptive) comparison of the following aspects of the two streams: i. Habitat (environmental parameters) (qualitatively in words or visually using appropriate tools, e.g., tables, histograms, etc.) ii. Taxa composition, i.e. compare the overall taxa lists (qualitatively in words or visually using appropriate tools, e.g., tables, histograms, etc.) b. Quantitative comparison of the biodiversity of the two streams using the recommended indices: i. Overall richness (quantitatively using Mann-Whitney U test, a non-parametric test for difference between the two streams) ii. Overall abundance (graphically using rank-abundance curve) (see diagram in Fig. 3)

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iii. Overall diversity (quantitatively using Mann-Whitney U test of Shannon index scores ) c. Quantitative comparison of the quality of the two streams using the recommended index: i. SingScore (quantitatively using Mann-Whitney U test of SingScores) Notes: Presentation of results: The Results section should be a summary of your results and NOT contain all the raw data that you collected (raw data such as tables from your practical schedule as well as taxa lists should instead be in an appendix). It should also NOT consist of only tables and graphs without any text. Remember, text comes before the table/figure. Statistical analysis: Keep your statistical analysis simple. You do NOT have to perform a test for normalitywe will assume for this exercise that the sampling results are NOT normally distributed. Therefore you should use a non-parametric statistical tool to test for differences between the streams; in this case, you are only required to use Mann-Whitney U test.

5) Discussion, including the following subsections: a. Clear and specific answers to your specific research questions, i.e. whether or not your hypotheses are supported by the results, and what factors might be influencing the similarity or difference in the various aspects between the two streams? Note: You are only expected to qualitatively discuss any possible associations between biological data and environmental parameters. You are NOT required to do any quantitative test for this such as correlation, regression, or multivariate statistics. b. Brief qualitative comparison of the two sampling methods (Surber sampler and tray net) based on the respective taxa lists obtained by each method c. A separate subsection with answers to all the Discussion Questions, which are highlighted in bold and italics earlier in this practical schedule (four answers in total). The deadline for submission of the hard copy of your report is 5 pm, Monday, 10 Oct 2011. Late submissions will be penalized (10% deduction of the report mark for each working day past deadline). This exercise will count towards 20% of your final mark for this module.

Abundance

Commonest Fig. 3. Axes for a rank-abundance curve

Abundance rank

Rarest

REFERENCES
Blakely, T. J., J. S. Harding, E. Clews & M. J. Winterbourn, 2010. An illustrated guide to the freshwater macroinvertebrates of Singapore. School of Biological Sciences, University of Canterbury. 74 pp. Lim, K. K. P. & P. K. L. Ng, 1990. A Guide to the Freshwater Fishes of Singapore. Singapore Science Centre, Singapore. 160 pp.

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