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Development of an Index of Trophic. Completeness for benthic macroinvertebrate communities in flowing waters. Hydrobiologica 427: 135-141. Pearson, R. G. ...
BENTHIC MACROINVERTEBRATE COMMUNITY COMPOSITION IN HIGH ALTITUDE ANDEAN STREAMS

Name: Michel Asselman (0571822) Project: MSc research (50 EC) Supervisor: drs. R. Loayza-Muro Examiner: Prof. dr. W. Admiraal Date: 14 September 2009 – 28 May 2010 Program: Limnology and Oceanography University of Amsterdam IBED - AEE  

 

TABLE OF CONTENTS Abstract

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Part I: Family Composition and Functional Feeding Group Assemblage as a Result of High UVB radiation and Metal Pollution Introduction Study Area Materials and Methods Physicochemical Measurements Family Richness and Trophic Diversity Results Physiochemical Measurements Family Richness and Trophic Diversty Discussion Family Composition and Diversity Conclusions Acknowledgements

4 4 5 6 6 6 7 7 8 11 11 13 13

Part II: Species identification using the mitochondrial COI gene Introduction Materials and Methods Macroinvertebrate Sampling DNA Extraction COI Amplification Purification and Sequencing Alignment and Analysis Results Discussion Conclusions Acknowledgements

14 14 15 15 15 15 15 16 17 18 19 19

References

20

APPENDIX A

25

APPENDIX B

32

APPENDIX C

33

APPENDIX D

34

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ABSTRACT Stressful conditions in high altitude (>3000m ASL) Andean streams are generated by elevated UVB radiation and metal pollution, which challenge the adaptation and survival of aquatic fauna. However, the species composition of benthic macroinvertebrate communities is underexplored and the impact of stress factors has not been specified. Therefore, macroinvertebrates were collected at four stream sites during the dry and wet season, identified to the lowest taxonomic level possible and categorized under five functional feeding groups (Part 1). Furthermore the fauna of selected taxonomic groups from the sites was genetically analyzed to try and verify their taxonomic status (Part 2). It has been found that UVB and metal pollution, alone or in combination, may cause alterations in benthic macroinvertebrate community composition through a decrease of organisms abundance and taxa richness, a replacement of sensitive taxa by more tolerant ones, and a strong shift in the diversity of functional feeding groups. Moreover, since few studies have addressed the identification of macroinvertebrate species in this region and the current taxonomical keys do not allow a low identity resolution, the COI gene was used to assess species identification and community composition. A total of 366 animals were selected from three major families (Baetidae, Gammaridae and Simuliidae) present at the reference sites. After amplification of the COI gene, the 192 best samples were sequenced, aligned and Neighbor-Joining trees were constructed. A total of 13 new species of Baetidae, a new Gammaridae species and a new Simuliidae species were distinguished because none of the animals collected in this study showed a match with species known in Genbank, indicating that all animals collected in this work had not been sequenced before. Therefore, morphological identification and genetic analysis should be merged together in order to fully assess the macroinvertebrate community composition in high altitude streams in the Peruvian Cordillera Blanca.

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PART I: EFFECTS OF HIGH UVB AND METAL POLLUTION ON BENTHIC COMMUNITY AND FUNCTIONAL FEEDING GROUP ASSEMBLAGE INTRODUCTION The Earth is facing climate changes, which include increasing levels of ultraviolet-B radiation (UVB; λ=280-315 nm). Even though UVB is a small component of the sun’s total emitted energy, much research has been performed to understand its effects on aquatic ecosystems (Bothwell et al., 1994; Williamson, 1995; Kiffney et al., 1997a,b; Vinebrooke and Leavitt, 1999; Clare, 2000; Kelly et al., 2003). These effects include the reduction of macroinvertebrate biomass (Kelly et al., 2003), modification of community composition through the replacement of species (Bothwell et al., 1994; Kiffney et al., 1997a) and alteration of the trophic structure (Bothwell et al., 1994; Cooke and Williamson, 2006). However, less is known about the interaction of UVB with other stressors such as metals (Liess et al., 2001; Duquesne and Liess, 2003; Zuellig et al., 2007). Metal pollution produced by natural leaching or human activities such as mining exploitation can have direct and indirect effects on benthic organisms, thus representing a major factor shaping community composition (Kiffney and Clements, 1996; Clements et al., 2000). Direct effects involve a reduction of the abundance and richness of sensitive taxa such as mayflies (Ephemeroptera), stoneflies (Plecoptera), blackflies (Simuliidae) and caddishflies (Trichoptera) compared to more metal tolerant taxa, such as midges (Chironomidae), beetles (Coleoptera) and worms (Oligochaeta) (Armitage et al., 1983; Cota et al., 2002). Indirect effects are mainly alteration of food sources, which limit food quality and availability (Pavluk et al., 2000). The impact of environmental disturbances on benthic communities may be assessed not only on their diversity, but also from a functional perspective (Rosenberg and Resh, 1993; Schreibler and Debandi, 2008). Evaluating functional feeding group (FFG) composition (Cummins, 1995) can reveal alterations of trophic relationships within a community, which may in turn affect the whole aquatic ecosystem (Merritt et al., 1996, 2002; Cummins et al., 2005). An appropriate approach to study these effects is the evaluation of FFGs replacement or turnover between sites (Ward et al., 1999; Amoros and Bornette, 2002) according to their sensitivity to stressors such as UV-B radiation and metals. High altitude Andean streams show elevated UVB, combined with low oxygen concentrations and low water temperature, which may be considered extreme for stream biota (Sommaruga, 2001; Jacobsen, 2007). Environmental disturbances such as elevated metal pollution may add further stress to macroinvertebrates inhabiting high altitude streams, although this has been scarcely explored. Therefore, the aim of the study was to the effects of UVB and metals on the benthic macroinvertebrate community composition in high altitude streams. For this purpose we evaluated the physical and chemical properties of streams, as well as the composition of macroinvertebrate communities at reference and polluted sites located at 3000 and 4000m above sea level (ASL) during the wet and dry season in the Cordillera Blanca (Peruvian Andes).

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STUDY AREA For this research a reference and a metal polluted site were chosen at 3000m ASL (low altitude) in the city of Huaraz and at 4000m ASL (high altitude) in the Huascaran National Park within the Cordillera Blanca (Peruvian Andes). At low altitude the reference site was located in the Casca River, and the was located in the Quilcayhuanca River, which is the major water supply for the city of Huaraz. Metal pollution in the Quilcayhuanca River originates mainly from natural leaching and is visible as reddish iron precipitates in the riverbed substrate. At high altitude the reference site was located in an unnamed spring and the polluted site was located, upstream from the low polluted site, in the Quilcayhuanca River (Figure 1). Both sites were located in the Huascaran National Park, for which special permission for this study was needed to enter. In the park, grassland was the dominant vegetation and Incan shepherds were present with their cattle.

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MATERIALS & METHODS PHYSICOCHEMICAL MEASUREMENTS Water physicochemical parameters were measured with WTW Multi 3400i Multi-Parameter, equipped with a SenTix® 41-3 sensor for pH, CellOx 325-3 sensor for dissolved oxygen and TetraCon 325-3 sensor for conductivity. Water transparency was measured using a 120cm polycarbonate turbidity tube. Flow was measured using a flow meter and discharge was calculated as the average of the three products of mean velocity, mean depth and stream width at three crosssections. Measurements of UVB were done using a Delta Ohm hand-held HD 2302 photo radiometer meter with a LP 471 UVB probe (meter model; UVB detector). Water samples were taken and analyzed for metals, phosphate and ammonium by SGS del Peru S.A.C. (Lima, Peru).

FAMILY RICHNESS AND TROPHIC DIVERSITY Benthic macroinvertebrates were collected using the “kick-sampling” method where a net was kept, at the stream’s bottom, downstream of a person who disturbs the sediment. This method was repeated on different types of microhabitats at each site to have a representative sample of the macroinvertebrate community. Macroinvertebrates were identified to family level using North American taxonomic keys (Merritt and Cummins, 1996), after which abundance and family richness were calculated. To assess the turnover of community composition between reference and polluted sites, low and high altitude sites, and over seasons, the Whittaker index was used (Formula 1). An outcome of “0” means that two sites share the same community composition whereas an outcome of “1” means that they differ completely.

Formula 1: Whittaker index, in which β= the turnover, in which, S= the total number of species recorded in both communities and α=average number of species found within the communities.

To evaluate the trophic completeness and functionality of communities the families present were categorized under five FFGs according to Merritt and Cummins (1996), i.e. gathering collectors, filtering collectors, predators, scrapers and shredders.

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RESULTS PHYSICOCHEMICAL MEASUREMENTS Maximum UVB was higher at high altitude sites and during the dry season compared to low sites and the wet season. At the polluted sites conductivity was higher, and pH lower than at reference sites, especially during the dry season. No differences in phosphates and nitrogenous compounds were observed and slight fluctuations were observed for dissolved oxygen concentrations. Discharge and water flow were higher during the wet season, while transparency was lower (Table 1). Metal concentrations were higher, and exceeded the MPC, at the polluted sites than at reference sites at both altitudes and seasons (Table 2). Table 1: Chemical and physical parameters at four sites in the Cordillera Blanca, Peru.

Wet

Season Altitude Low High

High

Reference

1.03

29

7.3

10

0.5

0.01

5.9

26

640

69

Polluted Reference Polluted Reference Polluted Reference

1.03 2.95 2.95 2.01 2.01 4.89

125 52 179 73 155 70

4.6 7.5 4.2 6.5 5.2 7.4

10.9 8.7 12.4 14 14.2 13.4

0.5 0.5 0.5 0.5 0.5 0.5

0.01 0.01 0.01 0.01 0.01 0.01

5.8 6.0 5.1 5.2 5.4 5.6

35 120 74 41 64 120

800 17 740 420 550 15

73 6 80 43 50 5

Polluted

4.89

219

4.3

14.1

0.5

0.01

5.5

120

460

56

Table 2: Concentrations of metals (mg/L) and MPC values at four sites in the Cordillera Blanca, Peru. A “-“ means no data available.

Wet

Season Altitude Low High Low Dry

Dry

Low

UVB Ndissolved water conductivity Temp phosphates transparency discharge max pH ammonia oxygen flow (uS/cm) (°C) (mg/L) (cm) (L/s) 2 (W/m ) (mg/L) (mg/L) (cm/s)

SITE

High

SITE

Al

As

Ca

Cd

Co

Cu

Fe

Mn

Ni

Sr

Zn

Reference

1.31

0.005

2.9

0.001

0.002

0.003

1.4

0.039

0.003

0.020

0.012

Polluted Reference Polluted Reference Polluted Reference

1.43 0.01 1.61 0.045 1.35 0.045

0.005 0.005 0.005 0.005 0.006 0.005

8.8 6.9 11.5 6.2 12.5 9.2

0.001 0.001 0.001 0.001 0.001 0.001

0.011 0.001 0.014 0.001 0.012 0.001

0.004 0.003 0.007 0.003 0.006 0.003

1.7 0.1 2.7 0.3 0.6 0.1

0.431 0.002 0.618 0.020 0.491 0.002

0.018 0.001 0.024 0.001 0.021 0.001

0.040 0.021 0.046 0.051 0.062 0.030

0.131 0.005 0.152 0.005 0.152 0.005

Polluted

1.65

0.008

15.2

0.001

0.015

0.007

0.7

0.698

0.026

0.065

0.176

-

0.032

-

0.002

0.003

0.004

-

-

0.006

-

0.040

MPC

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FAMILY RICHNESS AND TROPHIC DIVERSITY In this study a total of 4954 animals and 31 families were found, with Diptera (9 families), Coleoptera (7 families) and Trichoptera (4 families) being the best represented orders. Most animals were collected during the dry season in the reference low site and least animals were collected during the wet season in the polluted low site. The highest number of families was observed in the reference high site during the dry season, the lowest number of families in the polluted low site during the wet season. Further, trophic diversity (FFG) was higher at reference sites compared to polluted sites (Table 3). Table 3: Number of families, number individuals and functional feeding groups (FFG) at four sites in the Cordillera Blanca Peru. Wet Season Low altitude High altitude Reference

Polluted

#  Families  

10  

#  individuals  

228   4  

#  FFG  

Low altitude Reference

Dry Season High altitude

Reference

Polluted

Polluted

Reference

Polluted

2  

13  

13  

17  

3  

22  

10  

4  

913  

78  

2376  

92  

931  

332  

2  

5  

3  

5  

2  

5  

4  

Differences in family composition were observed between reference and polluted sites, low and high altitude, and wet and dry season (Table 4). Chironomidae (Diptera) was the only family present at all sites throughout the year. Gammaridae (Amphipoda) were most abundant at the reference high altitude during the wet and dry season, while Baetidae (Ephemeroptera) were most abundant at the reference and polluted low sites during the wet and dry season, respectively. The Whittaker index showed that the highest turnover in community composition was between the polluted low and high sites during the dry season (β = 0.846). The two most similar communities were those from the reference low and high sites during the dry season (β = 0.333). Pollution yielded the highest turnover between the reference and polluted low sites during the wet season (β = 0.833), and the least turnover between the reference and polluted high sites during the wet season (β = 0.462). Seasonality caused a low turnover at the reference low site (β = 0.407) and a higher turnover at the polluted low site (β = 0.600) (Table 5). At all sites, gathering collectors were more abundant during the dry season than during the wet season. At the reference sites, filter feeders were more abundant at low sites, whereas shredders were more abundant at high sites. At the polluted high site, filter feeders were only present during the dry season (Figure 2).

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Table 4: Relative family abundance (%) at four sites in the Cordillera Blanca, Peru. Ref = reference, Pol = polluted.

Order

Wet season Low altitude High altitude Ref Pol Ref Pol

Family

Acariformes Amphipoda Annelida Annelida Coleoptera

50.00

Gammaridae Hirudinea Oligochaeta Dysticidae Elmidae adult Elmidae larvae Scirtidae Stapheilinidae Hypogastruridae Isotomidae Sminthuridae Crustacea Copepoda Ostracoda Diptera Blepharaceridae Ceratopogonidae Chironomidae Dixidae Empididae Simuliidae Tabanidae Tipulidae Psychodidae Ephemeroptera Baetidae Heptagenidae Homoptera Aphididae Nematoda Dugesia Trichoptera Hydrobiosidae Hydroptiliidae Leptoceridae Limniphilidae Turbellaria # collected animals

0.33 70.54 0.33

5.13 1.28 2.56 8.97 1.28

9.65 0.33 5.37 3.83 3.95

Dry season Low altitude High altitude Ref Pol Ref Pol 1.53

4.40 38.78

0.11 3.87 0.32 0.11

0.04 0.04

0.11

11.18

3.85 3.85 1.28

0.32 0.11

7.56

0.21 5.05

0.32

0.11 26.21 4.30 0.21

0.32 71.88 12.46 0.96 0.32

0.04 10.53 0.44 2.63 32.46

50.00

0.88

46.15 20.51 2.56

0.11

87.31 0.04 1.96 2.98

32.61

0.11 0.11 37.72 0.44

0.04

10.08

1.28

0.22 0.88 1.32 228

0.22

1.28

913

78

4

2.56 0.26

65.22

0.64 0.04 0.09 1.79 0.21 0.43 2348

2.17

92

0.11 8.06 0.11 0.11 0.11 0.21 0.11

1.92

1.83 5.48 931

0.32 313

Table 5: Whittaker index for four sites in the Cordillera Blanca, Peru. Reference vs Polluted Wet

Low vs High

Dry

Wet

Low

High

Low

High

0.833

0.462

0.700

0.500

Wet vs Dry Season Dry

Low

High

Reference Polluted Reference Polluted Reference Polluted Reference Polluted 0.652

0.733

0.333

0.846

0.407

0.600

0.429

  9  

0.478

Figure 2: Composition of the functional feeding groups at four sites in the Cordillera Blanca, Peru.

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DISCUSSION PHYSICOCHEMICAL MEASUREMENTS At all polluted sites, cobalt, copper, nickel and zinc exceeded the maximal permitted concentration (MPC) or risk level (RIVM, 2008) and hence can be considered having toxic effects on aquatic biota. The MPC establishes values above which negative effects on benthic macroinvertebrates and thus community composition are expected. Since metal mixtures are likely to be present in the polluted streams sampled here, this alteration could be higher due to metals acting additively. Another additional negative effect could be represented by metals present in the sediment (Hare, 1992), although not measured in this study. The low pH found at the polluted sites, most likely due to natural acid drainage, may contribute to the solubility and bioavailability of metal ionic forms, thus increasing the toxicity of dissolved metals (De Jonge et al., 2008). On the other hand, since low abundance and richness were found during the wet season compared to the dry season, current velocity and discharge may also have a negative effect on community composition (Mathuriau et al., 2008).

FAMILY COMPOSITION AND DIVERSITY Polluted sites at both altitudes and seasons showed the lowest number of individuals and families, most likely because of sensitivity of taxa to metals, as previously reported by Clements et al. (2000). A reduction in abundance in acid/metal polluted waters has been previously observed for Ephemeroptera (Otto and Svensson, 1983; Ökland and Ökland, 1986), Trichoptera (Scullion and Edwards, 1980) and Simuliidae (Kozlov et al., 2005), whereas Coleoptera (Gerhardt et al., 2004), Chironomidae (Wickham et al., 1987; De Haas et al., 2005) and Oligochaeta (De Jonge et al., 2008) showed to be persistent, as observed in the present study. Increased UVB radiation at high altitude reference sites did not result in lowered richness compared to the low altitude reference sites, thus it seems unlikely that animals, at high altitude, have a higher cost of maintenance due to repair of UVB induced damage (Congdon et al., 2000). The increased family richness at the high altitude polluted site compared to the low altitude polluted site during both seasons, however, might be due to the presence of macroinvertebrates with increased pigment levels at the high altitude site (Merritt, pers. comm.). These pigments that are produced by macroinvertebrates living at high altitude are used as metal chelators, and thereby protect the animals against elevated metal concentrations (Szpoganicz et al., 2002). However, the opposite has also been proposed, where adaptation to one stressor may increase the sensitivity to a second one (Wilson, 1988), thus, organisms previously exposed UVB have a lowered tolerance against metal pollution and visa versa (Kashian et al., 2004, 2007). Nevertheless, the presence of UVB and metals, solely or together, induced a shift in the composition of benthic macroinvertebrate communities. The absence of scrapers and the low abundance of shredders in metal polluted sites could be explained by the presence of metal hydroxide precipitation layers on the stream substrate (Gerhardt et al., 2004). These groups are considered specialized feeders, and are most abundant in clean streams (Cummins and Klug, 1979) . Collector feeders, on the other hand, are generalists with a broader range of food sources, and thus should be more tolerant to pollution with altering food quality (Pavluk et al., 2000). However, the absence and low abundance of filtering collectors at the polluted high site during the dry season may indicate that concentrations of   11  

dissolved metals were toxic to these organisms. Spatial and temporal variability of the discharge and current velocity regulate the accumulation and distribution of litter and particulate matter (Pearson et al., 1989; Dudgeon and Bretschko, 1996), with higher accumulation during the dry season. (Covich, 1988; Larned, 2000). These changes in food composition may cause an increased abundance of gathering collectors and a reduction of filtering collectors during the dry season (Pavluk et al., 2000). Also, low stream velocity has been reported to have a positive correlation with macroalgae growth (Branco et al., 2005), which may explain the presence of shredders at the reference high site. Since predators, scrapers and shredders have a more mobile behavior, due to active food searching, streams with higher current velocity are thought be a less suitable habitat for these groups of organisms (Tamanova et al., 2006).

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CONCLUSION Altitude (3000m vs. 4000 m) and metal pollution alters community composition in high altitude Andean streams. This effect was the strongest under combined stressor conditions at the high altitude polluted site. Strong declines were observed in taxa described elsewhere as sensitive and at the same time tolerant taxa increased. These changes could be caused by direct effects on the invertebrates species differing in sensitivity to UV-B and the metal mixture at polluted sites. However, the representation of the functional feeding groups at high altitude and polluted sites changed compared to low altitude, clean sites, and this was attributed to indirect effect resulting from a changed food availability and food quality. Acknowledgements – I would like to thank BSc. C. P. Merritt, BSc. J. Marticorena-Ruiz and Lic. R. Elias-Letts are gratefully thanked for their assistance during the laboratory work. Also dr. Julio Palomino is thanked for his assistance in selecting suitable locations and his help during the sampling in the field

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PART II: BENTHIC MACROINVERTEBRATE SPECIES IDENTIFICATION USING THE MITOCHONDRIAL COI GENE INTRODUCTION High altitude Andes represent a unique ecoregion showing elevated UV-B radiation, and low oxygen water concentrations and low water temperatures in streams (Sommaruga, 2001; Jacobsen 2007). These natural environmental conditions may be considered extreme for stream biota, thus making worth exploring the presence of particular specialized fauna. In this region little is known about the diversity of benthic macroinvertebrates (Tomanova et al., 2006) and current knowledge mostly refers to the family level since available taxonomical keys do not allow lower resolution. Present keys have been developed for low altitude areas of South America (Roldán, 1996; Fernández and Domínguez, 2001) and North America (Merritt and Cummins, 1996), which may bring confounding information when identifying organisms from Andean high altitude regions. For this reason, a different approach should be used to identify organisms at a genus or species level, and obtain a higher resolution for describing benthic macroinvertebrate populations and communities in this region. DNA barcoding genes, such as mitochondrial oxidase sub-units I and II (COI and COII), have been extensively used to solve phylogenetic and taxonomic problems at multiple hierarchical levels in insects; from closely related species (Sperling and Hickney, 1994; Beckenback et al., 1993; Brower, 1996) to genera (Frati et al., 1997), families (Howland and Hewitt, 1995), and orders (Liu and Beckenback, 1992). Therefore, the aim of the present study was to use of the COI gene to determine the identity of species from the most representative macroinvertebrate families in Andean streams in the Cordillera Blanca (Peru), and relate it to previous findings on effects of UV-B radiation and metal pollution described in Part I.

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MATERIALS AND METHODS MACROINVERTEBRATE SAMPLING Sampling was performed at the reference low and high sites (Part I) during the wet season. Animals were gently removed from small stones surface from the riverbed with a soft toothbrush, collecting in small jars and transported with absolute ethanol (Sigma-Aldrich Chemie, Zwijndrecht, Netherlands) to the Laboratory of Ecotoxicology at the Universidad Cayetano Heredia, in Lima, Peru. In addition, algal masses were collected at the reference high site. In the laboratory, animals were separated by family according to Merritt and Cummins (1996), stored in 1.5mL Eppendorf tubes containing 1.0mL absolute ethanol and stored at -70ºC until transported to the University of Amsterdam, Netherlands, at ambient temperature.

DNA EXTRACTION A selection of 366 animals, belonging to three families, Baetidae (Ephemeroptera) from 3000m ASL and 4000m ASL, Gammaridae (Amphipoda) from 4000m ASL and Simuliidae (Diptera) from 3000m ASL, was made. Pictures of every sample were taken before DNA extraction using an Olympus Camedia C-5060 wide zoom camera mounted on an Olympus SZ11 stereomicroscope and the program AnalySIS 5.0 (Soft Imaging System GmBH, 2004). DNA was extracted using the DNeasy Blood & Tissue Kit (Catalog No. 69504, Qiagen, Venlo, Netherlands). Organisms were placed in a 1.0mL microcentrifuge tube with 180µl ATL buffer and zirconia beads (1.0mm) and homogenized using a Precellys Tissue Homogenizer (program 6000_2x35_15). Then, 20µL proteinase K was added and samples were mixed thoroughly using a vortex and incubated for 60 minutes at 56°C. After incubation, 200µL preheated AL buffer was added, mixed by vortexing, and followed by an addition of 200µl absolute ethanol and mixed again thoroughly. The samples were then centrifuged for 30 seconds at 3000rpm and the whole mixture was pipetted, including precipitate, into a DNeasy Mini spin column placed in a 2mL collection tube, incubated at room temperature for 15 minutes and centrifuged at 8000rpm for 1 minute, after which the flow-through was discarded. Then 500µL AW1 buffer was added and centrifuged for 1 minute at 8000rpm, after which the flowthrough was discarded. Then 500µL AW2 buffer was added and centrifuged at 14000rpm for 3 minutes. The flow-through and collection tube were discarded and the DNeasy Mini spin column was placed in a new 1.5mL microcentrifuge tube, 50µL pre-heated (56°C) AE buffer was added and incubated at 56°C for 10 minutes. After incubation, the mixture was centrifuged at 8000rpm for 1 minute. The presence of DNA was verified using a 1% agarose gel, and the quantity and quality of the DNA extract was measured using a NanoDrop spectrophotometer (APPENDIX A). DNA extracts were then stored at -20°C.

COI AMPLIFICATION DNA samples were taken from -20°C and defrosted on ice and, if necessary, diluted to a maximum concentration of 35ng/µL. An approximate 700-basepair portion of the COI gene was amplified using a Polymerase Chain Reaction (PCR) and the primers LCO1490 and HCO2198 (Folmer et al., 1994). For this, 2.5µL of the extracted template DNA was mixed with 17.5µL of a PCR cocktail by pipetting the mixture up and down twice, vortexing and centrifuging it briefly. This PCR cocktail consisted of H2O (8.3µL), PCR Buffer (4.0µL; 5X), deoxyNucleotide TriPhosphates (dNTP) (4.0µL; 1mM), a forward and reverse primer (0.50µL; 10µM each; Folmer et   15  

al., 1994; Table 6) and “Hot Start” taq polymerase (0.20µL; 5u/µL). PCR cycling conditions were: initial denaturation at 98°C for 30 seconds, 35 cycles of denaturation at 98°C for 5 seconds, annealing at 48°C for 5 seconds, extension at 72°C for 15 sec and thereafter a final extension step at 72°C for 60 seconds and a cool down at 4°C for 300 seconds To visualize and verify the purity of the achieved the PCR product the samples were loaded on a 1% agarose gel and run in an electrophoresis bath. Table 6: LCO1490 (forward) and HCO2198 (reverse) primers. Direction Name Sequence Forward LCO1490 5'-GGTCAACAAATCATAAAGATATTGG-3' Reverse HCO2198 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

PURIFICATION AND SEQUENCING For purification and sequencing, 15µl of the PCR amplified COI samples were sent to MacroGen Europe (Amsterdam, Netherlands).

ALIGNMENT AND ANALYSIS COI sequences were aligned using Genious 4.8 for Macintosh (Drummond et al., 2009). The same program was used to find population DNA barcoding sets of the families that were analyzed in Genbank (Table 7). A Neighbor-Joining tree was constructed using the Tamura-Nei distance model and a 70% similarity cost matrix. Table 7: Genbank Index numbers of the population set DNA sequences used to construct Neighbor-Joining trees. Family Baetidae Gammaridae Simuliidae

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Genbank Index 37529676 161170193 225381168

 

RESULTS From 220 samples amplified by PCR, the 192 showing the highest quality bands on a 1% agarose gel were selected for sequencing. The length of the sequences ranged from 615 base pairs to 838 base pairs with a mean of 707 base pairs (Table 8). A total of 15 different groups were distinguished, whereas the low altitude Baetidae were most diverse and the Simuliidae and Gammaridae were least diverse. Table 8: Number of DNA samples, COI gene amplification, sequenced PCR products, average number of base pairs ± standard deviation (SD) and number of distinctive groups per family. Family Baetidae Baetidae Gammaridae Simmulliidae Total

DNA altitude extracted low high high low

COI amplified

# base Sequenced pairs

± SD

# groups found

96 94 96 80

88 31 85 16

77 30 70 15

695 695 719 707

± ± ± ±

28 26 38 29

9 6 1 1

366

220

192

707 ±

35

15

Variation within the thirteen different groups of Baetidae ranged between 0.3% and 2.9%, and variation between groups was at least 7% (Appendix B). Further only in two groups samples of both the low and high altitude sites were present. Intraspecific variation found within the group of Gammaridae was 0.6% (Appendix C), and for Simuliidae this was 1.1% (Appendix D).

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DISCUSSION Although DNA could be extracted from all samples, a PCR product was only observed in 60% of the samples. The primers used in this study were developed for invertebrates (Folmer et al., 1994), and have been used previously in studies with Baetidae (Ball et al., 2005; Ståhls and Savolainen, 2008), Simuliidae (Rivera and Currie, 2009) and Gammaridae (Witt et al., 2006), and are therefore suitable for the samples used in this study. A possible explanation for the lack of amplified PCR product in 40% of the samples could be due to bad cycling conditions, however, this is not a valid explanation. This because in each PCR run, some samples had amplified PCR product. Another explanation for the lack of PCR product in several samples could be because of low DNA quality at the time of extraction and amplification. The thirteen groups of Baetidae observed in the Neighbor-Joining tree can be considered different species because the variation between all groups exceeded the previously recorded intra-specific variation of up to 6.6% (Ball et al., 2005). Groups were not assigned species names, since not all groups had a previously sequenced species within the intra specific variation range, or had multiple species within this range. The group of amphipods can be considered one species since the variation within this group was less than the previously recorded intra specific variation of 3.8% (Witt et al., 2006). The genus that was closest related to the samples in the Neighbor-Joining tree was Hyalella, which has been previously recorded at high altitudes, above 3500m ASL, in the Chilean Andes (González and Watling, 2001,2002,2003). Therefore it would be likely that the samples from the present study are also from the Hyalella genus. The single group of Simuliidae could be considered one species since the variation within this group is less than the 6.5% previously recorded by Rivera and Currie (2009). Where the closest species known had a variation of approximately 20%, nothing could be said about a possible species or genus name. New taxonomical keys for South America (Fernández and Domínguez, 2009), have an updated list of species, however, the geographical distribution is only limited to the country in which species were observed. Therefore, future research should focus on developing keys, with the use of present taxonomical literature and molecular tools, for more specific regions of South America.

18  

 

CONCLUSIONS The primers used to amplify the COI gene (LCO1490 and HCO2198) are suitable to detect new species of Baetidae, Amphipoda and Simulidae so far not incorporated in Genbank. Thirteen new genotypes of Baetidae were found underlining the need for further taxonomic analysis of the Andean mayflies., Simuliidae and Gammaridae populations each consist of only one species. So far it is indicated that Baetidae populations at low altitude have a higher species richness than at high altitude and this difference might be caused by altitude related selection stress tolerance. Acknowledgements – I would like to thank BSc. C. P. Merritt, BSc. L. Dong, A. M. H. Voetdijk, P. Kuperus and dr. J. A. H. Breeuwer are greatfully thanked for their assistance during the laborory work. Also dr. Julio Palomino and BSc. J. Marticorena-Ruiz are thanked for assistance in selecting suitable locations and help during the sampling in the field.

  19  

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APPENDIX A: Samples processed within Genetic Population Analysis. With the obtaines DNA concentration, DNA purity (260/230 > 0.7 is good)) and DNA quality (260/280 > 1.70 is good), the DNA concentration used in the COI PCR and the quality of the PCR product (0= no band, 1 is vague band, 2 is clear band, 1s/2s = with smear). Order Family

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band

Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph

B301 B302 B303 B304 B305 B306 B307 B308 B309 B310 B311 B312 B313 B314 B315 B316 B317 B318 B319 B320 B321 B322 B323 B324 B325 B326 B327 B328 B329 B330 B331 B332 B333 B334 B335 B336 B337 B338 B339 B340 B341 B342 B343 B344 B345 B346 B347 B348 B349

Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae

3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000

0.12 0.02 0.07 0.04 0.10 0.07 0.03 0.05 0.03 0.06 0.03 0.05 0.03 0.10 0.02 0.02 0.02 0.03 0.02 0.04 0.18 0.02 0.15 0.19 0.02 0.10 0.01 0.03 0.03 0.02 0.02 0.05 0.02 0.02 0.03 0.06 0.04 0.08 0.08 0.01 0.01 0.04 0.01 0.05 0.03 0.01 0.01 0.01 0.05

1.72 0.81 1.71 0.79 1.62 0.56 0.63 0.72 0.50 0.99 1.04 1.10 0.89 1.24 1.30 0.91 0.75 0.94 0.95 1.48 1.71 1.20 1.36 1.56 1.57 1.22 1.18 1.17 1.10 1.29 1.81 1.66 2.71 2.14 2.44 2.70 1.62 1.98 1.97 1.10 1.97 1.55 2.05 2.01 2.20 1.91 2.48 2.66 2.13

12.4 2.2 12.2 3.5 3.2 2.5 1.5 3.0 0.9 2.8 2.0 2.8 2.8 4.2 3.0 2.7 1.6 3.3 2.4 6.2 9.4 3.7 5.4 3.7 5.2 5.9 2.4 4.9 8.7 5.8 9.8 18.4 8.8 8.7 16.6 17.4 10.4 33.9 30.9 2.7 3.5 19.8 4.0 23.9 7.7 3.0 3.2 5.2 23.9

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 1

12.4 2.2 12.2 3.5 3.2 2.5 1.5 3.0 0.9 2.8 2.0 2.8 2.8 4.2 3.0 2.7 1.6 3.3 2.4 6.2 9.4 3.7 5.4 3.7 5.2 5.9 2.4 4.9 8.7 5.8 9.8 18.4 8.8 8.7 16.6 17.4 10.4 33.9 30.9 2.7 3.5 19.8 4.0 23.9 7.7 3.0 3.2 5.2 23.9

2 1 2 1 2s 1 0 1 1 2s 1 1s 1 2s 1 1 0 2s 0 2s 2s 2s 2 2s 2 2 0 0 1 1 1 2 0 2s 2s 1 1 1s 2s 2 0 1s 2s 1s 1s 1 1 2s 1

  25  

Order Family

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band

Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph

B350 B351 B352 B353 B354 B355 B356 B357 B358 B359 B360 B361 B362 B363 B364 B365 B366 B367 B368 B369 B370 B371 B372 B373 B374 B375 B376 B377 B378 B379 B380 B381 B382 B383 B384 B385 B386 B387 B388 B389 B390 B391 B392 B393 B394 B395 B396 B401 B402 B403 B404 B405 B406 B407

26  

Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae

3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 4000 4000 4000 4000 4000 4000 4000

0.09 0.18 0.30 0.09 0.14 0.01 0.23 0.07 0.16 0.27 0.14 0.23 0.04 0.09 0.19 0.10 0.08 0.49 0.17 0.31 0.29 0.17 0.09 0.46 0.17 0.43 0.35 0.28 0.34 0.19 0.18 0.10 0.08 0.21 0.14 0.12 0.07 0.14 0.11 0.60 0.05 0.10 0.12 0.15 0.12 0.04 0.30 0.10 0.10 0.38 0.75 0.15 0.13 0.08

2.07 2.11 2.12 2.14 2.13 2.00 2.09 1.76 2.12 2.02 2.12 2.15 2.08 1.97 2.09 2.04 1.83 2.11 2.04 2.08 2.07 2.11 2.18 2.19 1.17 2.17 2.21 2.05 2.07 2.18 2.04 1.98 2.04 2.22 2.10 2.20 2.07 2.28 2.16 2.16 1.87 2.11 2.03 2.06 2.03 1.87 2.13 2.07 2.15 2.17 2.08 2.19 2.14 2.07

44.3 69.0 157.0 46.5 65.5 2.7 73.3 31.5 76.5 30.1 54.1 69.3 20.0 47.4 66.8 36.3 42.9 294.0 82.0 131.7 112.2 67.5 42.3 240.1 62.5 145.2 124.0 106.4 150.8 59.7 89.1 22.0 17.8 95.5 54.8 54.9 24.2 21.8 18.4 132.0 14.7 34.6 37.0 27.7 44.4 19.5 68.7 27.4 41.6 100.9 400.2 54.4 61.3 27.8

2 3 6 2 3 1 3 1 3 1 2 3 3 2 3 1 2 10 4 5 4 3 2 10 3 6 5 4 6 3 3 1 1 4 3 3 1 1 1 5 1 1 2 1 2 1 3 1 2 4 15 2 2 1

22.2 23.0 26.2 23.3 21.8 2.7 24.4 31.5 25.5 30.1 27.1 23.1 6.7 23.7 22.3 36.3 21.5 29.4 20.5 26.3 28.1 22.5 21.2 24.0 20.8 24.2 24.8 26.6 25.1 19.9 29.7 22.0 17.8 23.9 18.3 18.3 24.2 21.8 18.4 26.4 14.7 34.6 18.5 27.7 22.2 19.5 22.9 27.4 20.8 25.2 26.7 27.2 30.7 27.8

2 2 2 2 2 0 2 2 2 1 2 1 2 2 1 2 2 2 2 2 2 2 2s 2 2s 1s 1s 2 2s 1s 2s 2 2 2s 2 1 2s 1 1 1 1 2s 2s 1 2s 1 2 0 0 0 1s 0 0 0

 

Order Family

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band

Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph

B408 B409 B410 B411 B412 B413 B414 B415 B416 B417 B418 B419 B420 B421 B422 B423 B424 B425 B426 B427 B428 B429 B430 B431 B432 B433 B434 B435 B436 B437 B438 B439 B440 B441 B442 B443 B444 B445 B446 B447 B448 B449 B450 B451 B452 B453 B454 B455 B456 B457 B458 B459 B460 B461

Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae

4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000

0.34 0.64 0.21 0.29 0.19 0.08 0.07 0.09 0.47 2.00 0.22 0.15 0.19 0.21 0.10 0.19 0.14 0.09 0.05 0.12 0.14 0.13 0.13 0.15 0.04 0.06 0.20 0.07 0.19 0.25 0.10 0.08 0.04 0.09 0.18 0.05 0.07 0.06 0.12 0.17 0.06 0.45 0.26 0.14 0.09 0.15 0.08 0.05 0.07 0.12 0.04 0.06 0.11 0.13

2.15 2.16 2.14 2.18 2.21 2.15 1.77 1.66 1.84 0.69 1.38 1.81 1.97 2.01 1.79 2.16 1.99 2.22 1.86 2.27 2.26 2.08 2.10 2.11 2.40 2.02 2.19 2.12 2.19 2.13 2.22 1.77 2.29 2.23 2.19 2.40 2.02 1.97 2.04 2.13 2.08 2.21 2.17 2.19 2.01 2.32 2.08 2.19 2.05 2.23 2.63 2.00 2.11 2.04

98.4 126.2 105.9 61.4 50.1 39.9 38.4 41.3 188.3 164.2 80.3 75.3 102.9 113.1 46.9 76.4 75.1 45.3 25.1 50.4 73.1 68.4 56.5 74.5 26.9 28.8 101.0 37.4 94.7 136.7 52.0 33.3 16.5 38.1 72.4 26.6 36.4 30.1 62.6 92.5 28.9 171.6 108.8 37.7 30.3 50.2 32.0 20.7 31.3 36.0 19.4 28.3 24.4 37.3

4 5 4 2 2 2 2 2 7 6 3 3 4 4 2 3 3 2 1 2 3 3 2 3 1 1 4 2 4 5 2 1 1 2 3 1 2 1 2 3 1 6 4 2 1 2 1 1 1 2 1 1 1 2

24.6 25.2 26.5 30.7 25.1 20.0 19.2 20.7 26.9 27.4 26.8 25.1 25.7 28.3 23.5 25.5 25.0 22.7 25.1 25.2 24.4 22.8 28.3 24.8 26.9 28.8 25.3 18.7 23.7 27.3 26.0 33.3 16.5 19.1 24.1 26.6 18.2 30.1 31.3 30.8 28.9 28.6 27.2 18.9 30.3 25.1 32.0 20.7 31.3 18.0 19.4 28.3 24.4 18.7

0 1s 1s 0 0 1s 1s 2s 1s 1s 1 2 1 1 1 1 1 1 1 2 0 2s 1 1 1s 0 1 2 2 2 0 0 2s 2s 0 1s 1s 2s 0 0 0 1s 1s 2s 0 2s 1s 1s 1s 1s 0 0 2s 1s

  27  

Order Family

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band

Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Eph Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

B462 B463 B464 B465 B466 B467 B468 B469 B470 B471 B472 B473 B474 B475 B476 B477 B478 B479 B480 B481 B482 B483 B484 B485 B486 B487 B488 B489 B490 B491 B492 B493 B494 GA01 GA02 GA03 GA04 GA05 GA06 GA07 GA08 GA09 GA10 GA11 GA12 GA13 GA14 GA15 GA16 GA17 GA18 GA19 GA20 GA21

28  

Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Baetidae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae

4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000

0.19 0.08 0.05 0.06 0.33 0.13 0.10 0.05 0.05 0.10 0.03 0.08 0.10 0.11 0.32 0.16 0.26 0.35 0.49 0.15 0.20 0.08 0.05 0.05 0.07 0.08 0.17 0.11 0.83 0.09 0.52 0.14 0.13 0.21 0.42 0.1 0.33 0.13 0.29 0.19 0.13 0.75 0.23 0.15 0.27 0.12 0.15 0.18 0.15 0.13 0.23 0.09 0.03 0.14

1.58 1.77 1.99 2.15 2.24 2.15 2.00 2.28 2.08 1.95 1.89 2.23 1.97 1.95 1.56 1.79 2.04 2.04 1.87 2.11 1.69 2.24 2.10 1.47 1.98 2.11 2.03 2.07 1.55 2.04 2.14 1.90 2.04 2.14 2.12 2 2.11 2.11 2.21 2.04 1.79 1.65 2.02 2.06 ? 1.96 1.98 1.99 2.23 2.02 2.13 1.89 1.59 2.05

99.4 23.9 26.2 26.5 50.8 61.1 45.5 24.9 18.9 27.9 15.8 36.3 34.0 44.3 133.8 59.6 112.1 118.2 251.0 52.6 76.6 38.7 16.5 10.2 32.6 26.8 54.0 57.7 45.6 44.9 246.5 68.6 50.4 77.8 95.4 41.8 97.3 48.7 52.2 86.2 56.5 64.7 89 68.3 72.9 47.7 60.7 63.1 83.2 37.9 29.6 31.2 13.1 57.8

4 1 1 1 2 2 2 1 1 1 1 2 2 2 5 2 4 4 9 2 3 2 1 1 1 1 2 2 2 2 9 3 2 3 4 2 4 2 2 3 2 3 3 3 3 2 2 2 3 2 1 1 1 2

24.9 23.9 26.2 26.5 25.4 30.6 22.8 24.9 18.9 27.9 15.8 18.2 17.0 22.2 26.8 29.8 28.0 29.6 27.9 26.3 25.5 19.4 16.5 10.2 32.6 26.8 27.0 28.9 22.8 22.5 27.4 22.9 25.2 25.9 23.9 20.9 24.3 24.4 26.1 28.7 28.3 21.6 29.7 22.8 24.3 23.9 30.4 31.6 27.7 19.0 29.6 31.2 13.1 28.9

0 1s 1s 0 0 1s 0 0 0 0 0 0 0 1s 0 0 2s 1s 2s 0 0 0 0 0 1s 1s 2s 1s 0 1s 2s 0 0 2 2 2 2 2s 2s 2s 2s 0 2s 0 1s 2s 2s 2s 2s 0 0 2s 2s 2s

 

Order Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

Family Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band GA22 4000 0.11 2.03 38.4 2 19.2 2s GA23 4000 0.18 1.98 40.8 2 20.4 2s GA24 4000 0.14 2.09 63.6 2 31.8 2s GA25 4000 0.16 2.28 106.5 4 26.6 2s GA26 4000 0.09 2.1 32.9 1 32.9 2s GA27 4000 0.27 2.13 82.7 3 27.6 2s GA28 4000 0.07 1.89 19.1 1 19.1 2s GA29 4000 0.07 1.71 26.2 1 26.2 2s GA30 4000 0.13 1.8 22.1 1 22.1 2s GA31 4000 0.05 1.51 22 1 22.0 1 GA32 4000 0.09 1.96 23.7 1 23.7 2s GA33 4000 0.25 2.11 106.1 4 26.5 0 GA34 4000 0.19 1.55 40.2 2 20.1 2s GA35 4000 0.04 1.71 15.4 1 15.4 0 GA36 4000 0.25 1.96 123.2 5 24.6 1s GA37 4000 0.16 1.94 53.1 2 26.6 0 GA38 4000 0.34 2.12 158.3 6 26.4 2s GA39 4000 0.13 2.04 54.4 2 27.2 2s GA40 4000 0.26 2.11 99.2 4 24.8 1s GA41 4000 0.32 2.12 117.9 4 29.5 1s GA42 4000 0.23 2.06 75 3 25.0 2s GA43 4000 0.38 2.14 157.9 6 26.3 2s GA44 4000 0.25 2.03 113.8 4 28.5 2s GA45 4000 0.4 2.12 144.5 5 28.9 2s GA46 4000 0.17 1.96 39.5 2 19.8 2s GA47 4000 0.23 2.07 94.5 3 31.5 1s GA48 4000 0.02 1.6 5.5 1 5.5 0 GA49 4000 0.13 2.02 52.3 2 26.2 2 GA50 4000 0.07 1.48 19 1 19.0 2 GA51 4000 0.04 1.15 16.5 1 16.5 2 GA52 4000 0.06 1.85 10.9 1 10.9 2 GA53 4000 0.03 1.57 8.5 1 8.5 2s GA54 4000 0.43 2.05 60.7 2 30.4 2 GA55 4000 0.19 2.02 68.1 3 22.7 2s GA56 4000 0.16 1.45 27.9 1 27.9 2s GA57 4000 0.04 2 13.1 1 13.1 2s GA58 4000 0.07 1.74 21.9 1 21.9 2 GA59 4000 0.04 1.54 11.9 1 11.9 2s GA60 4000 0.04 1.76 16.5 1 16.5 2s GA61 4000 0.01 2.36 5.5 1 5.5 0 GA62 4000 0.02 1.71 6.1 1 6.1 1s GA63 4000 0.01 1.35 3 1 3.0 0 GA64 4000 0.04 1.81 13.2 1 13.2 2s GA65 4000 0.03 1.65 11.2 1 11.2 2s GA66 4000 0.02 2.09 7.4 1 7.4 2s GA67 4000 0.02 1.63 6.7 1 6.7 2s GA68 4000 0.03 1.34 3.2 1 3.2 2s GA69 4000 0.05 2.02 15.9 1 15.9 2s GA70 4000 0.05 1.69 15.6 1 15.6 2s GA71 4000 0.13 1.82 20.2 1 20.2 2s GA72 4000 0.05 1.34 17.9 1 17.9 1s GA73 4000 0.07 1.8 23 1 23.0 2s GA74 4000 0.09 1.79 29.6 1 29.6 2 GA75 4000 0.12 1.98 36.3 2 18.2 2s

  29  

Order Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip

30  

Family Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Gamaridae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band GA76 4000 0.1 1.79 33.6 2 16.8 2s GA77 4000 0.11 1.5 42.5 2 21.3 2s GA78 4000 0.1 2.1 43.3 2 21.7 2s GA79 4000 0.42 2.04 80.9 3 27.0 2s GA80 4000 1.93 2.08 142.2 5 28.4 2s GA81 4000 0.3 2.1 78.8 3 26.3 2s GA82 4000 0.22 2.07 59.6 2 29.8 2 GA83 4000 0.08 2.15 35.7 2 17.9 2s GA84 4000 0.12 1.73 39.1 2 19.6 2s GA85 4000 0.18 2.09 72.4 3 24.1 0 GA86 4000 0.27 2.04 77 3 25.7 2 GA87 4000 0.16 2.06 20.6 1 20.6 2 GA88 4000 0.31 2.05 29.2 1 29.2 2 GA89 4000 0.5 2.13 95.7 4 23.9 2 GA90 4000 0.14 2.14 37.9 2 19.0 2 GA91 4000 0.14 2.1 52.8 2 26.4 2 GA92 4000 0.23 2.18 59.4 2 29.7 2s GA93 4000 0.28 2.14 63.7 2 31.9 1 GA94 4000 0.97 2.05 52.7 2 26.4 2s GA95 4000 0.47 2.11 60.7 2 30.4 2s GA96 4000 0.19 2.07 64.1 3 21.4 2s S01 3000 0.63 2.19 339.6 12 28.3 2 S02 3000 0.18 2.28 77.6 3 25.9 0 S03 3000 0.16 2.31 54.1 2 27.1 0 S04 3000 0.27 2.22 132.4 5 26.5 0 S05 3000 0.26 2.28 86.2 3 28.7 0 S06 3000 0.25 2.33 74.7 3 24.9 0 S07 3000 0.19 2.04 69.2 3 23.1 0 S08 3000 0.23 2.27 97.6 4 24.4 0 S09 3000 0.4 1.83 149.3 5 29.9 0 S10 3000 0.09 2.18 35.1 2 17.6 0 S11 3000 0.18 2.15 80.9 3 27.0 1 S12 3000 0.07 2.42 22.9 1 22.9 0 S13 3000 0.32 2.24 125.5 5 25.1 0 S14 3000 0.27 2.26 89.5 3 29.8 0 S15 3000 0.56 2.19 162.7 6 27.1 2s S16 3000 0.18 2.19 77.3 3 25.8 0 S17 3000 0.15 2.24 59 2 29.5 0 S18 3000 0.31 2.38 62.3 2 31.2 0 S19 3000 0.66 2.27 103.8 4 26.0 0 S20 3000 0.12 2.64 33.6 2 16.8 0 S21 3000 0.07 3.04 28.2 1 28.2 0 S22 3000 0.25 2.33 114.7 4 28.7 2 S23 3000 0.18 1.99 83 3 27.7 0 S24 3000 0.28 2.35 37.2 2 18.6 0 S25 3000 0.33 2.06 73.2 3 24.4 0 S26 3000 0.3 2.28 53.8 2 26.9 0 S27 3000 0.13 2.6 64.9 3 21.6 0 S28 3000 0.47 2.31 119.8 4 30.0 0 S29 3000 0.06 3.19 22.4 1 22.4 0 S30 3000 0.19 2.58 29.7 1 29.7 0 S31 3000 0.17 2.23 41 2 20.5 0 S32 3000 0.13 2.32 53.8 2 26.9 0 S33 3000 0.12 2.39 37.9 2 19.0 0

 

Order Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip Dip

Family Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae Simulidae

Sample Altitude 260/230 260/280 DNA ng/ul Dilution [DNA] PCR COI band S34 3000 8.12 1.4 89.4 3 29.8 0 S35 3000 0.31 2.31 122.5 4 30.6 0 S36 3000 0.23 2.32 109.1 4 27.3 0 S37 3000 0.15 2.2 70 3 23.3 0 S38 3000 0.04 3.11 16.3 1 16.3 0 S39 3000 0.19 2.59 30.7 1 30.7 0 S40 3000 0.09 2.45 36.2 2 18.1 0 S41 3000 0.37 2.15 108 4 27.0 0 S42 3000 0.18 2.2 79.4 3 26.5 0 S43 3000 0.42 2.12 123.5 4 30.9 0 S44 3000 1.08 2.16 194.4 7 27.8 2 S45 3000 0.18 1.75 81.5 3 27.2 0 S46 3000 0.17 2.14 83.7 3 27.9 0 S47 3000 0.13 2.12 57.3 2 28.7 0 S48 3000 0.66 2.15 292.4 10 29.2 2s S49 3000 0.8 2.15 419.8 14 30.0 2s S50 3000 0.93 2.19 133.7 5 26.7 0 S51 3000 0.29 2.12 87.9 3 29.3 0 S52 3000 0.33 2.15 108.9 4 27.2 2s S53 3000 0.31 2.18 126 5 25.2 0 S54 3000 0.3 2.07 91.9 3 30.6 0 S55 3000 0.15 1.89 67.6 3 22.5 0 S56 3000 0.13 2.13 51.6 2 25.8 0 S57 3000 0.13 2.04 37.3 2 18.7 0 S58 3000 0.65 2.06 220.9 8 27.6 2s S59 3000 1 2.19 219.3 8 27.4 2s S60 3000 0.15 2.17 33.9 2 17.0 0 S61 3000 0.31 2.18 146.5 5 29.3 2s S62 3000 0.34 2.14 143.1 5 28.6 2s S63 3000 0.17 2.04 79.2 3 26.4 0 S64 3000 0.29 1.89 75.5 3 25.2 0 S65 3000 0.11 2.04 45.7 2 22.9 0 S66 3000 0.15 1.79 71.8 3 23.9 0 S67 3000 0.16 2 53.7 2 26.9 0 S68 3000 0.31 1.82 139.5 5 27.9 0 S69 3000 0.17 1.84 68 3 22.7 0 S70 3000 0.17 1.65 74.6 3 24.9 1s S71 3000 0.21 1.95 94.4 4 23.6 2s S72 3000 0.1 1.88 48.4 2 24.2 0 S73 3000 0.41 2.04 222.1 8 27.8 0 S74 3000 0.36 2.09 166.1 6 27.7 2 S75 3000 0.11 1.78 47.3 2 23.7 0 S76 3000 0.09 1.68 43.2 2 21.6 0 S77 3000 0.09 1.81 25.1 1 25.1 2s S78 3000 0.51 2.13 163.3 6 27.2 2s S79 3000 0.19 2.12 42.7 2 21.4 0 S80 3000 0.08 2.04 39.7 2 19.9 0

  31