Disrupting the biodiversity – ecosystem function relationship: response of shredders and leaf breakdown to urbanization in Andean streams

Urbanization is a major driver of stream ecosystems impairment and often associated with multiple stressors and species loss. A challenge is to understand how those stressors alter the relationship between biodiversity and ecosystem functioning (B-EF). In Andean streams of southern Ecuador, we assessed the response of shredder diversity and organic matter breakdown (OMB) to urbanization and identified the urban-associated stressors disrupting the B-EF relationship. A leaf-litter bag experiment during stable flow conditions in 2016 was carried out to quantify total OMB and shredder-mediated OMB, which was estimated to represent the B-EF relationship. We calculated the taxonomic and functional diversity of shredder invertebrates associated with leaf packs. Also, a suite of physicochemical and habitat stressors was weekly measured during the field experiment. Along the urbanization gradient, both taxonomic and functional diversity of shredders declined while OMB rates decayed. Shredders were absent and their contribution to OMB was null at the most urbanized sites. The B-EF relationship was interrupted through nutrient enrichment and physical habitat homogenization as a consequence of urbanization. These results demonstrate how species loss propagates to ecosystem functions in urbanized streams and how environmental stressors alter the B-EF relationship. Better land-use practices are crucial in Andean catchments to guarantee ecosystem services which are the result of a successful B-EF relationships.


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Introduction 30 Aquatic ecosystems contain high biodiversity, but at the same time, they evidence a faster species 31 loss than marine or terrestrial ecosystems (Collen et al., 2014; Sánchez-Bayo and Wyckhuys, 2019). 32 Unfortunately, species loss also implies a reduction of ecosystem functions (Cardinale et al., 2006). 33 Species diversity is crucial for the dynamics and functioning of ecosystems, for that reason the 34 relationship between biodiversity and ecosystem functioning (B-EF) has been well established and is 35 a central research issue (Loreau et al., 2001;Tilman et al., 2014). Also, the B-EF relationships can be 36 altered by environmental gradients, complicating the prediction of the effects of species loss in 37 ecosystems subjected to environmental stress (McKie et al., 2009). Therefore, it is imperative to 38 understand the mechanisms that interrupt this relationship trough environmental disturbance 39 gradients such as urbanization. 40 Urbanization is a complex process involving permanent changes in the landscape such as the 41 transformation of land use from rural (i.e. forest, agriculture) to urban (Antrop, 2004). Also,42 urbanization is related to numerous stressors that interact synergistically on streams (Walsh et al.,43 2005), affecting the hydrology, chemistry and aquatic communities via multiple pathways (Paul and 44 Meyer, 2001;Walsh et al., 2005). Urbanized streams often show a reduction in channel width and 45 substrate size of streambed due to sediment inputs, as well as an increase in nutrient concentrations 46 and other pollutants from sewage inputs and runoff (Allan, 2004). When these stream characteristics 47 are altered by urbanization there will be a consequence on the macroinvertebrate communities (Roy 48 et Macroinvertebrates can play important roles in stream ecosystem functioning (Wallace and Webster, 54 1996). For instance, organic matter breakdown (OMB) is mediated by shredder invertebrates, as well 55 as by leaching, microbial conditioning, and physical abrasion (Gessner et al., 1999). Indeed, 56 shredders importance on OMB has been well documented in different regions (Jonsson et  In this study, we used shredder diversity (taxonomic and functional diversity) and OMB rates to 89 quantify the effects of urbanization on stream integrity in an Andean catchment influenced by a rapid 90 and unmanaged metropolitan expansion. In particular, we examined (i) how shredder invertebrates 91 are affected by an urbanization gradient, (ii) whether OMB rates are similarly affected by the 92 urbanization gradient, and (iii) which environmental variables altered by urbanization are disturbing 93 the biodiversityecosystem function relationship (i.e. shredder-mediated OMB rates). We 94 hypothesized that the loss of shredder diversity because of urbanization is also propagated in the 95 ecosystem function in Andean streams. 96

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Material and Methods 97

Study area and sampling sites 98
This study was conducted in the city of Loja and its surrounding areas in the southern Ecuadorian 99 Andes (Figure 1) the native forests extend to the catchment divide. Also, a few small plots for farming and small pine 104 plantations can be found in the surroundings of the city. Historically, the central city area has been 105 located along the valley and has grown towards the outskirts. This urban sprawl has transformed land 106 cover around the city from forest to pasture and then to urban over decades leaving an urbanization 107 gradient within the catchment. Sewage is directly discharged to streams and agricultural and urban 108 runoff is not managed Therefore, we selected 12 independent streams along such urbanization 109 gradient, having three replicated streams at urban, pasture, forest mixed with pasture, and forest sites, 110 respectively. At each stream, we placed a sampling site consisted of a 30 m reach located 111 downstream of the target land cover.

Environmental variables 116
In the dry season of 2016 (September), during the period of stable flow conditions, a total of 16 117 environmental variables were measured (Table 1). Water temperature, conductivity, pH, and 118 dissolved oxygen were measured using a YSI 556 multi-probe meter (Yellow Springs, Ohio, USA); 119 whereas the concentration of dissolved inorganic nitrogen (DIN = nitrate + nitrite + ammonium), 120 dissolved reactive phosphorus (DRP), and turbidity were determined in laboratory using standard 121 methods (APHA, 2017). Channel width and depth, and current velocity were determined at four to 122 six points along each sampling site using a flowmeter FP311 (Global Water, California, USA). These 123 variables were measured four times in seven-day intervals over throughout the OMB experiment (see 124 below). Substrate size was estimated visually by randomly place 4 plots (1 × 1 m) at each sampling 125 site. The percent of silt, sand, gravel, pebble, cobble, boulder, bedrock was determined according to 126 the Wentworth scale to calculate a substrate index (Harding et al., 2009). 127 We quantified the percentage of land covered by native forest, pasture, crops, plantations, and 128 urbanization within each catchment of the 12 selected streams. Land cover was obtained upstream the 129 sampling sites using an open-access GIS and a land use map for 2016 obtained in another study 130 (Iñiguez-Armijos pers. comm.). In addition, we quantified riparian canopy cover (%) at four 131 equidistant points from hemispherical photographs taken at 1.3 m above the water surface. 132 Photographs were acquired with a digital camera equipped with a fish-eye lens and then processed in 133 Gap Light Analyzer Version 2.0. 134 Labill.). These species are frequent in the riparian zone of the studied streams, but only croton is a 143 native species whereas alder and eucalyptus have been planted. By using different litter types, we 144 considered potential variation in shredder colonization and breakdown rates. We enclosed 4 ± 0.05 g 145 of air-dried leaves in 15 × 15 cm coarse mesh bags (10 mm) to allow access by shredders. combusted (4 h/500°C), and reweighted leaf material to obtain ash-free dry mas (AFDM) (Bärlocher, 156 2005). An extra set of 5 leaf bags of each litter type was similarly treated to correct mass loss of the 157 final AFDM caused by handling (Bärlocher, 2005). 158 Over the 30 days, at each sampling site, we recorded water temperature in 1 h intervals using HOBO 159 pendant data loggers (Onset, Massachusetts, USA) placed within the mesh bags and secured to the 160 iron bars. This data was used to calculate the sum of degree days (dd -1 ) during the incubation period 161 to standardize breakdown rate (k) by temperature, and to reduce differences between stream types. 162 Then, breakdown rates were estimated using an exponential decay model per degree days (k dd -1 ), as 163 follows: 164 where 0 is initial air-dried mass, is final AFDM at time , and is incubation time in degree-166 days. Total breakdown rates (ktotal), which is breakdown in coarse mesh bags, were corrected for 167 microbial breakdown (kcoarsekfine) to calculate the contribution of shredders invertebrates (kshredders) 168 to this ecosystem process (McKie et al., 2009) (Table 1). 169

Shredder diversity 170
All invertebrates recovered from the coarse mesh bags were counted and identified to the lowest 171 taxonomic level (usually genus) using available literature for South America (Dominguez and  172 Fernández, 2009). Then, we assigned the invertebrates to a functional feeding group (Ramírez and  173 Gutiérrez-Fonseca, 2014). We only selected shredder invertebrates for this study to be directly 174 associated with leaf litter breakdown. We classified all shredder invertebrates according to three 175 biological traits (size, life cycle duration, respiration) and 14 different modalities based on literature 176 available (Tachet, 2000). Traits such as food, dietary preference, or reproduction were excluded to be 177 general for shredders in this study. 178 For each litter type, we calculated taxonomic richness (taxa per bag-1) and abundance (individuals 179 per bag-1) of shredders in R environment (R Development Core Team, 2019) using the 'vegan' and 180 'BiodiversityR' packages (see Table 1). Also, we estimated functional richness and Rao's quadratic 181 entropy (RaoQ) based on Gower's distances of our biological traits using the 'FD' package (Laliberté 182 et Legendre, 2010). Due to no shredder species were recorded in the most urbanized streams, for 187 practical and representation purposes we added zero values of functional diversity for these sites. 188

Urbanization gradient 189
We produced an urbanization gradient using the environmental variables indicated in Table 1  were build using the 'mgcv' package (Wood, 2017). 225

Shredder diversity and urbanization 227
A total of 287 shedder invertebrates belonging to nine genera of seven families and four orders were 228 collected across all sites. No shredders were found at urban sites, whereas the lowest shredder 229 richness was observed at pasture sites with seven taxa and the highest at mixed forest-pasture and 230 forest sites with eight taxa each. Moreover, mixed forest-pasture and forest sites had more individual 231 of shredder invertebrates that other sites. ZIM models ( Table 2), indicated that the effect of the 232 urbanization gradient was negative in both taxonomic richness and abundance of shredders ( Figure  233 2A and B). Urbanization also affected negatively the functional richness and RaoQ of shredder 234 invertebrates, but the response of functional diversity was a gradual decrease along the urbanization 235 gradient (Figure 2C and D).

Breakdown rates and urbanization 245
On average, ktotal was 0.0019 dd −1 and the contribution of shredders to OMB was much lower than 246 microorganisms (Table 1), indeed kshredders represented around 21% of total breakdown. GAM models 247 ( Table 3), indicated that OMB responded negatively along the urbanization gradient being faster in 248 forest sites diminishing towards urbanized sites ( Figure 3A). However, the response of OMB 249 mediated by shredders showed a drastic decrease at urban sites ( Figure 3B).

Urbanization and the biodiversity and ecosystem function relationship
After the selection process of environmental variables associated to the urban gradient (i.e. PC1) for 263 GAM modelling, DRP, pH, turbidity, and substrate size were retained. DIN, and dissolved oxygen 264 were highly correlated with DRP; whereas channel width and depth, and current velocity were highly 265 correlated with substrate size. We excluded urban land cover for being a variance-inflated variable. 266 GAM modeling indicated that DRP and substrate size were the most meaningful variables and had a 267 strong negative effect on kshredders, i.e. B-EF relationship (Figure 4), each explaining 39% and 41% of 268 the deviance explained, respectively (Table 3) Although the relationship of B-EF has been extensively studied (Tilman et al., 2014), it is still 277 necessary to understand how environmental disturbance gradients can alter this relationship. In this 278 study, we assessed the response of taxonomic and functional diversity of shredder invertebrates as 279 well as OMB rates along an urbanization gradient in Andean streams. We detected that the diversity 280 of shredder invertebrates decreases from natural to more urbanized sites and that shredders are totally 281 absent in urban streams. We also found that OMB rates are negatively affected along the urbanization 282 gradient slowing down this ecosystem process towards urban sites and that urban-associated 283 variables representing water chemistry (e.g. DRP) and physical habitat (e.g. substrate size) of the 284 streams are altering the relationship between shredders and OMB. 285 DRP is produced by phosphate inputs to stream ecosystems from wastewater coming from sewage 286 systems of the urban areas (Paul and Meyer, 2001). In our study, DRP concentrations were low at 287 forest sites but increased considerably towards urban sites (up to 25-fold greater). At the same time, 288 we observed a decrease in the taxonomic and functional diversity of shredder invertebrates and a 289 slowdown in OMB along the urbanization gradient. Although a moderate increase in the 290 concentration of nutrients such as P can stimulate OMB by shredders in temperate streams 291 (Woodward et al., 2012), in montane Andean streams the increasing of P has been shown to slow 292 down this OMB (Iñiguez-Armijos et al., 2016). Other stream parameters such as dissolved oxygen 293 and DIN were negative and positively associated with DRP. This correlation can be explained by the 294 same land-use intensification along the urbanization gradient, which facilitates the incorporation of 295 organic and inorganic pollutants that interact synergistically and reduce the stream ecosystem 296 integrity (Paul and Meyer, 2001). The increase in N and P concentrations from sewage (e.g. human 297 waste and detergents) decreases the dissolved oxygen (Allan, 2004). Nutrient enrichment favors 298 microbial activity which increases respiration rates and decreases the availability of oxygen in the 299 streams (Walsh et al., 2005). High nutrient concentrations and reduced dissolved oxygen combined 300 can interact reducing or eliminating shredder invertebrates (Couceiro et al., 2006;Iñiguez-Armijos et 301 al., 2016). This pattern is supported by our findings, but we also observed that the reduction in OMB 302 by shredders can be attributed to an abrupt loss of their taxonomic and functional diversity towards 303 more urbanized sites as an effect of nutrient enrichment and low availability of dissolved oxygen. 304 With regard to substrate size, riparian land-use change and bank erosion produce streambed 305 sedimentation in urbanized streams, reducing substrate size and its heterogeneity affecting negatively 306 the macroinvertebrate diversity (Couceiro et al., 2010). After the loss of macroinvertebrate taxa such 307 as shredders due to the alteration of substrate size and type, their ecological role will be also affected. 308 In our study, we found that the more the urbanization gradient increases, the less the substrate size 309 which is largely dominated by sand, silt and mud. This consequence can explain the strong negative 310 effect of substrate size on OMB mediated by shredders and their loss of taxonomic and functional 311 diversity. Size and type of the substrate condition the presence of several members of the 312 macroinvertebrate community in Andean stream ecosystems as substrate provides habitat, refuge and 313 food (Miserendino and Pizzolon, 2004). Therefore, as the heterogeneity and size of the substrate 314 decrease, there would be a loss taxonomic and functional diversity of shredders invertebrates, and 315 thus their contribution to OMB at the ecosystem level. Other hydraulic parameters such as channel 316 width and depth, and current velocity were negatively correlated to substrate size. In the Andes, 317 streams in natural areas show high current velocity and turbulent flow, they are wide and deep, the 318 streambed presents different substrate sizes and contains several shredder invertebrates taxa (Iñiguez-319 Armijos et al., 2018a; Vimos-Lojano et al., 2020). On the contrary, the studied streams at urban sites 320 presented a laminar flow as a consequence of sediment deposition in the streambed dominated by 321 small particle sizes such as silt and mud. Also, these streams were less wide due to enclosure, and 322 have been documented as lacking shredder invertebrates (Iñiguez-Armijos et al., 2016). The 323 alteration of these hydraulic variables, as we have described here, has negatively affected the OMB in 324 tropical streams (e.g. Martins et al., 2015); and in our case, we believe that it has resulted in the loss 325 of the taxonomic and functional diversity of shredder invertebrates as well as their capacity for 326 organic matter processing in Andean stream ecosystems. 327 Decades of research were needed to understand the leading role of biodiversity in the dynamics and 328 functioning of ecosystems, and to quantify the positive effect of species diversity on ecosystem 329 processes (Tilman et al., 2014). Macroinvertebrates play important roles in the functioning of stream 330 ecosystems, among others, shredder invertebrates contribute to the C cycle by feeding on CPOM 331 (Ramírez and Gutiérrez-Fonseca, 2014). However, anthropogenic environmental stress has 332 negatively affected the taxonomic and functional diversity of macroinvertebrates reflecting on the 333 ecosystem functioning of temperate streams (Voß and Schäfer, 2017). In our study on Andean 334 stream ecosystems, we observed that the urbanization gradient notably reduced both taxonomic and 335 functional diversity of shredders which also propagated to OMB rates mediated by this FFG. Indeed, 336 at the most urbanized sites, we did not find shredders indicating a null contribution of 337 macroinvertebrates on organic matter processing in urban streams in the Andes. 338 The importance of shredders in stream ecosystems is also given as they are key organisms for energy 339 transfer at the low levels of food webs (Ramírez and Gutiérrez-Fonseca, 2014; Merritt et al., 2017). 340 In montane streams the relevance of shredders can be even greater due to leaf litter is the major 341 energy source (Wipfli et al., 2007). When shredders reduce the size of CPOM, they allow other 342 organisms to have nutrients downstream as well as the accomplishment of other ecosystems 343 processes (Woodward, 2009;Vaughn, 2010;Benfield et al., 2017). Our findings suggest that there 344 could be an alteration in the transfer of organic C in urban Andean streams. Nevertheless, more 345 evidence is needed to understand the effect of shedders' loss on organic C fluxes in these tropical 346 mountain stream ecosystems. 347 We demonstrate that urbanization affected the B-EF relationships in Andean streams via nutrient 348 enrichment and substrate homogenization causing taxonomic and functional diversity loss of 349 shredders and OMB decline. The loss of keystone species mostly results in a reduction of ecosystem 350 functions (Cardinale et al., 2006;Vaughn, 2010). Also, species loss is associated with the loss of 351 functional biological traits related to ecosystem functioning (Flynn et al., 2009). However, it remains 352 to be understood how biological traits of shredders important for OMB are affected by stressors 353 associated with urbanization in Andean streams. Finally, by losing ecosystem functions, several 354 ecosystem services are also compromised. It is crucial to implement better land-use practices in 355 Andean catchments to guarantee environmental services at lower altitudes. 356 5