Accepted version of MS published in Freshwater Biology 2020 DOI: 10.1111/fwb.13472

Terrestrial–aquatic transitions: local abundances and movements of mature female are related to oviposition habits but not flight capability

Jill Lancaster1, Barbara J. Downes1 and Georgia K. Dwyer1,2

1 School of Geography, University of Melbourne, 221 Bouverie Street, Parkville, 3010, 2 Centre for Regional and Rural Futures, Deakin University, Locked Bag 20000, Geelong, Victoria 3220, Australia

Corresponding author: Jill Lancaster, School of Geography, University of Melbourne, 221 Bouverie Street, Parkville, Victoria 3010, Australia Email: [email protected]

RUNNINGHEAD: Terrestrial-aquatic transitions

KEYWORDS: aquatic , dispersal, flight, Trichoptera, wing morphology

Summary 1. Movement behaviours of adult aquatic can produce distinct spatial distribution patterns. Studies of adult abundance with distance away from water bodies are common and may invoke flight capability to explain species differences. In contrast, distribution patterns along river channels are poorly described, but are no less important for understanding population dynamics. Longitudinal patterns in adult abundance along short river lengths may differ between sexes and at different life stage transitions between aquatic and terrestrial environments, i.e. at emergence and oviposition. Flight capability is unlikely to influence longitudinal patterns created at emergence, but may influence local abundances of mature females seeking to lay eggs. We tested hypotheses about how local abundances of mature females might differ according to oviposition habits and flight capability. 2. We surveyed abundances of mature female caddisflies at adjacent riffle–pool pairs along short river lengths with homogenous riparian cover. Our survey included nine species in three families (, , ), which encompassed multiple different oviposition habits and a range of wing sizes and shapes. Several of the species oviposit preferentially in riffles. Accordingly, we tested for differences in female abundance between channel units (adjacent riffle–pool pairs). We also tested whether females attained higher abundances in some places along channels than others (i.e. over larger spatial scales and regardless of channel unit) which imply movements along the

1 channel and aggregation in some locations. Wing morphology was used as a proxy measure of flight capability and included measures on wing span, area, aspect ratio and the second moment of wing area. 3. Three distinctly different distribution patterns of mature female caddisflies were identified. The abundance of three species varied over larger scales only (multiple channel units). Six species that oviposit preferentially in riffles had higher female abundances at riffles than pools, but for only one did abundances also vary over larger scales. There was no association between these different patterns and measures of wing morphology, after removing metrics that were correlated and that differed systematically between taxonomic families. However, we could not reject the hypothesis that some aspect of flight behaviour may have contributed to observed patterns. 4. The diverse but distinct distributions of mature female caddisflies we observed along short channel lengths are novel and suggest that species differ in their propensity for movement along streams, which could have consequences for local densities of eggs and juveniles in the aquatic environment. The degree to which population sizes are coupled across the terrestrial-to-aquatic transition is rarely investigated in aquatic insects and may provide fresh insight into sources of spatial variation within populations. Similarly, a more nuanced approach to research on the flight of aquatic insects, including age- and sex- specific phenomena, may provide greater insight into the diverse ecological functions and consequences of movement.

1. INTRODUCTION

Key life history events for adult aquatic insects include emerging from the aquatic to terrestrial environments, mating, potentially dispersing between populations and, for females, depositing eggs or neonates back into the aquatic environment. These events are poorly understood for the vast majority of species. Even quite basic information about local distribution patterns of adults is scarce, in sharp contrast to a plethora of studies on the distribution of larvae within water bodies. The local distributions of terrestrial adults and aquatic juveniles may be correlated in some circumstances (Downes & Lancaster, 2018), and such associations may have consequences for population dynamics in both terrestrial and aquatic environments. At the transition from aquatic juveniles to terrestrial adults, for example, variations in benthic densities of larvae and pupae may correspond to local variations in the abundance of emerging adults, and the abundance of their terrestrial predators (Gray, 1993; Iwata, 2007). Similarly, at the transition from terrestrial to aquatic habitats, adults may aggregate at places where eggs are laid and produce local aggregation of juveniles, with potential consequences for spatial heterogeneity in consumer-resource dynamics for aquatic juveniles (Harrison & Hildrew, 2001). Thus, understanding sex- and age-specific distribution patterns of adults and how they arise, especially when life-cycle and habitat transitions are concurrent, may provide insights into how life history events affect population size (Hildrew et al., 2004; Encalada & Peckarsky, 2012; Lancaster & Downes, 2014).

2 Local distribution patterns of adults are likely to differ between the two aquatic–terrestrial habitat transitions (i.e. at emergence and oviposition) because adults of most species move away from water during maturation. Indeed, the final instar larvae of some stoneflies may travel long distances terrestrially before metamorphosing into adults (Kuusela & Huusko, 1996). Adult movements may range from short-distance movements within the immediate surroundings (Southwood, 1962), e.g. to avoid predators or locate resting sites, food and mates, to long-distance dispersal or migration (Anderson, 2009; Buden, 2010). The total distance travelled between emergence and oviposition will vary between species; some will travel multiple kilometres (Baldwin, West & Gomery, 1975; Masters et al., 2007) and even short-lived adults can move 10s to 100s of metres (e.g. Maciel-De-Freitas, Codeco & Lourenco-De-Oliveira, 2007). For many species, most individuals may spend their adult life within 50 m riparian corridors (Svensson, 1974; Petersen et al., 2004; Bogan & Boersma, 2012) but collective, short distance movements are sufficient to reduce the likelihood that an individual will emerge from and oviposit in the same location.

Regardless of where adult aquatic insects go or how far they fly, at some point females must locate water and find an oviposition site. Water bodies may be located via the polarization of light reflected off water surfaces (Kriska, Horváth & Andrikovics, 1998; Horváth & Varjú, 2004), whereas the suitability of a particular water body may depend on water-borne chemicals indicating the presence of predators or competitors (Resetarits Jr., 2001; Brodin, Johansson & Bergsten, 2006). Locating an oviposition site within a water body may be straightforward for species that broadcast eggs, but complex for those with specialized oviposition behaviours. Oviposition site selection can involve a hierarchy of decisions at multiple spatial scales (Hoffmann & Resh, 2003; Lancaster, Downes & Reich, 2003; Reich & Downes, 2003b) and this may involve a lot of flying. Thus, female distribution patterns may vary with oviposition habits, as well as their flight capability and behaviour. Many stream-dwelling insects, for example, oviposit primarily in particular channel units, e.g. riffles or pools (examples below), and thus must be capable and willing to fly along channels to locate a suitable riffle or pool. There is ample evidence that many aquatic insects fly along channels (Winterbourn et al., 2007; Graham, Storey & Smith, 2017), but how far individuals actually fly is largely unknown. Prolonged flight is risky, e.g. aerial predation can be significant (Nakano & Murakami, 2001; Fukui et al., 2006), so the odds of survival may be correlated with flight capability and behaviour, and related to species-specific movement patterns (Svensson, 1974).

The general aims of this study were to test hypotheses about the spatial distribution patterns of mature female caddisflies along short lengths of stream channels to elucidate how their local abundance patterns might be influenced by oviposition habits, flight capability and behaviour. Typically, newly emerged female caddisflies move away from water, mate and remain away from water during maturation (generally several weeks), before returning to water to oviposit (Svensson, 1972; Svensson, 1974). We know that some species oviposit preferentially in particular channel units. For example, some species lay eggs on submerged rocks in pools (Deutsch, 1984; Lancaster, Downes & Arnold, 2010b), whereas others oviposit in

3 riffles and primarily on rocks that protrude above the water surface, i.e. emergent rocks (Hoffmann & Resh, 2003; Reich & Downes, 2003a; Lancaster, Downes & Arnold, 2010b; Lancaster & Glaister, 2019). However, numbers of such emergent rocks vary between riffles and there is often a correlation of oviposition site densities with egg mass densities, and sometime with larval densities also (Lancaster & Downes, 2014; 2018). These correlations imply that females may fly along channels to search for places where oviposition sites are abundant, but little is known about such movements. Movements of adult aquatic insects are difficult to observe, especially for species that are predominantly nocturnal, but some inferences about movement are possible with appropriately designed surveys of abundance patterns.

We present one such survey here. Three main concepts underpin our empirical tests and inferences regarding how oviposition habits and flight might influence distribution patterns of mature females. First, we assume that females move independently through the riparian zone and could arrive, with equal probability, at any point along the water's edge for short river lengths (<1 km) with uniform riparian cover. Analogously, newly emerged females typically move away from the stream channel individually, not en masse. Once at the river, most mature females are likely to move at least a short distance along the channel to locate places that offer suitable oviposition sites. Such movements should create aggregations in these places; in the absence of longitudinal movement, spatial structure (i.e. high variance) in local abundances is unlikely, provided the riparian environment does not vary greatly along the stream. Second, mature females may aggregate where oviposition sites are abundant, which may correspond to geomorphic channel units such as riffles vs pools (see above). Female aggregations are unlikely to correspond to channel units for species with quite different oviposition behaviours, e.g. eggs that are laid terrestrially, broadcast over the water surface, or deposited along stream margins (Lancaster & Downes, 2013). Third, species-specific flight characteristics may determine how far individuals fly along channels and where aggregations occur. Females that are weak or unwilling fliers are likely to remain close to where they exited the riparian zone, perhaps travelling the distance of one or two channel units at most. Conversely, strong and willing fliers may range over long channel lengths (e.g. across multiple channel units), with the potential to accumulate in certain places, e.g. with landscape features or where oviposition sites are particularly good.

These three concepts can be combined to predict four types of distribution pattern for mature females over short river lengths. Local abundances of females may: vary between riffles and pools; vary over larger scales (i.e. abundance varies in different places along channel lengths); vary between both channel units and over larger scales; or show no patterns. For multiple species of caddisfly on short river lengths (450 m) with continuous riparian forest, we tested whether local abundances of mature females fit one of these four patterns. Samples of adult caddisflies from adjacent riffles and pools were used to test whether numbers of mature females were (i) higher at riffles than pools and (ii) correlated between adjacent riffle–pool pairs. This latter pattern indicates that some places have more females than others, regardless of channel unit, and suggests variations over larger scales. Additionally, we contrasted distribution patterns of species that

4 oviposit exclusively in riffles, against species with different oviposition habits. Finally, the study species also varied in wing size and shape, allowing us to use wing morphology to test whether flight capability was related to abundance patterns.

The combinations of significant vs non-significant outcomes of the two tests above lead to different interpretations regarding how patterns arise, as shown in Table 1. Pattern A, abundance patterns at larger scales only (river lengths with multiple channel units), is expected for species where oviposition sites are not associated with particular channel units, but flight capability and propensity are strong, and thus some places have more females than others because they respond to channel features other than riffles or pools. If oviposition occurs preferentially in particular types of channel unit then we expect abundances to differ accordingly. Weak fliers should fit Pattern B if most individuals fly no farther than the nearest suitable oviposition site, whereas stronger fliers should fit Pattern C if females inspect multiple channel units before selecting an oviposition site. Pattern D is expected for species that are weak fliers and oviposition is not associated with channel units. The flapping flight of insects is complex, but some morphological characters of wings can provide proxy measures of flight capability (Ellington, 1984a; Dudley, 2000). We measured two gross parameters (wing span and area) and two shape parameters (wing aspect ratio and the second moment of wing area). Unfortunately, we had no means to assess aspects of flight behaviour (e.g. flight propensity), but we acknowledge explicitly that multiple factors can influence flight distances. If species that are strong fliers (as determined by wing morphology) fit Patterns A and C, and weak fliers fit Patterns B and D, then it is impossible to determine whether flight capability and/or some aspect of behaviour are the primary underlying factors. However, if the data supported all patterns except predictions about flight capability, then this is consistent with the notion that flight behaviour is a potential underlying factor.

2. METHODS

2.1 Study sites and species Surveys were carried out primarily on the Little River, Victoria, Australia. Some samples were collected initially on the nearby Steavenson River, but these sites were abandoned subsequently due to vandalism. These rivers drain into the and subsequently into the . In the collection areas (Little River: S 37.345, E 145.751 280 m ASL; Steavenson River: S 37.488, E 145.752, 374 m ASL), the rivers had rocky substrates, gentle gradients and cool water temperatures (for details see Lancaster et al., in press) and largely intact and diverse riparian vegetation characterised by species of Eucalyptus, Acacia, Pomaderris and Dicksonia.

Nine species of caddisfly in three families were sufficiently abundant for statistical analyses: five taxa of Hydrobiosidae (Apsilochorema spp., Ethochorema turbidum (Neboiss), Taschorema evansi (Mosely), Ulmerochorema seona (Mosely), U. rubiconum (Neboiss)), two species of Hydropsychidae

5 (Cheumatopsyche AV3, Asmicridea edwardsi (McLachlan)), and two species of Leptoceridae (Notalina bifaria Neboiss, Triplectides proximus Neboiss). Two species of Apsilochorema were present in low numbers (A. gisbum (Mosely) and A. obliquum (Mosely)) and were combined together for analyses. Adults of these two species are morphologically similar and have similar oviposition habits (Lancaster et al., in press). There are at least three distinctly different oviposition habits within this set of nine species. The five hydrobiosids and one hydropsychid, Cheumatopsyche AV3, lay their eggs as plaque-shaped masses attached to the underside of emergent rocks in riffles (Reich & Downes, 2003a; Reich, 2004; Lancaster & Glaister, 2019). The second hydropsychid, Asmicridea edwardsi, also oviposit plaque-shaped masses attached to rocks in riffles, but on both submerged and emergent rocks (Reich, 2004). In contrast, of the two leptocerids, T. proximus produce spherical, gelatinous masses of eggs (St Clair, 1993) that may be released when females alight on the water surface or at the water's edge. Egg masses of this type can aggregate in shallow, slowly flowing water on the stream margins (pers. obs.). To our knowledge, the oviposition habits of Notalina spp. are undescribed, but they do not carry egg masses at the tip of the abdomen as do many other leptocerids (St Clair, 1993). Unfortunately, the pupation and emergence habits of these species have not yet been described.

2.2 Survey design and protocols Adults were collected in light traps during three surveys carried out over four consecutive nights in each of three summer months, January, February and March 2018. The first survey (January) used sites on both the Steavenson and Little Rivers and the following two (February, March) were carried out in the Little River only. The design of our light traps is established (Downes et al., 2017) and each trap comprised a 12 Volt battery, a timer and an ultraviolet fluorescent tube (6 Watt, 225 mm long tube), which was laid across a white tray (28 ´ 22 ´ 5 cm) with 70% ethanol to a depth of approx. 1.5 cm. This assembly was placed inside a black tub (diameter = 39 cm; height = 32 cm) to ensure that light did not spill sideways but was directed upwards. Thus, insects were not attracted from long distances away and only insects flying directly above the light would be collected. To ensure we collected individuals that were flying above the water, traps were placed either right at the water's edge or perched on rocks and logs within the channel. The positions of traps did not differ systematically between riffles and pools. Traps were deployed for 6 hours each night, beginning 15 minutes before sunset. Samples were collected each morning. Specimens were identified following (Neboiss, 1986; 1992) and a subset of mature females (~50%) were examined to assess ovarian maturation.

In each month and on each of four nights, light traps were placed at five pools and a riffle adjacent to each pool (a total of 10 traps). The exception was that only three pool–riffle pairs were sampled on the Little River in January, owing to previously vandalized equipment. All riffles and pools were within a 450 m stream length. Riffles above and below each pool were sampled on alternate nights, to ensure that insects flying both up- and downstream were sampled with equal effort. Channel units were replicates in our analyses not sample nights, so samples for each pool, and the riffles adjacent to each pool, were composited

6 over the four nights. Data for each channel unit were expressed as the mean number of trapped per night. The range of pool lengths was 16–30 m (mean = 22.8, SD = 6.8 m) and distances between sampled pools ranged from 20 to 100 m. Preliminary analyses indicated that pool and riffle lengths were not important variables in any analyses and will not be discussed further. Emergent rock densities were broadly similar across riffles (2.9–3.9 m-2) and the variation was much smaller than the order of magnitude range typically required to detect associations between densities of emergent rocks and egg masses (Lancaster, Downes & Arnold, 2010a; Lancaster & Downes, 2018).

2.3 Wing morphology The measures of wing morphology and their calculation are detailed elsewhere (Lancaster & Downes, 2017), so a brief description is provided here. Our analysis focused on two first-order descriptions of size, wing area and wing length (or wing span), and on two unitless, second order descriptions of shape, wing aspect ratio

(AR) and the non-dimensional radius of the second moment of wing area, �̂ (�). In general, lift forces and flight capability increase with wing size. In terms of wing shape, high AR reflects slender wing shapes, which are associated with power economy and extended flight, whereas broad wings have a low AR, which favours slow, agile flight and manoeuvrability (Betts & Wooton, 1988; Dudley, 2000; Cespedes, Penz &

DeVries, 2015). The second moment of wing area is a strong indicator of lift force. Values of �̂ (�) are low for wings that have broad bases and narrow tips and values increase as the broadest part of the wing shifts towards the tip. Wings with very broad tips and high �̂ (�) may confer agility and maneuvrability, but also increase the energetic power required for flight (Ellington, 1984b). Conversely, wings with lower values of

�̂ (�) (broad bases, or leading and trailing edges that are approximately parallel) may be better suited for extended or long-distance flight.

One pair of fore and hind wings were removed from each insect, dry mounted on a microscope slide and a digital image produced. Wings were oriented so that the forewing span was horizontal and perpendicular to the longitudinal axis of the insect body and the hind wing was oriented in the coupled position. Not all caddisflies have physically coupled wings (Stocks, 2010a; Stocks, 2010b), but this arrangement ensured that measurements were consistent across species. Wing measurements were carried out on digital images of coupled wing pairs in planform (the orientation of wings during the down stroke and the generation of lift forces) and using the software ImageJ 1.49s (Rasband, 1997–2012). There were 10 replicates for each species/sex combination in all but one case.

2.4 Statistical analyses To test whether numbers of mature females of each species differed between types of channel unit, we used a split-plot ANOVA in which the subject was Time (Jan., Feb., March; N=3) and Blocks, nested within Time, were adjacent pool–riffle pairs. Treatment (riffle vs pool) and the Treatment ´ Time interaction were tested

7 within subjects. This model allows the January subject to include data from riffle–pool pairs in both the Steavenson and Little Rivers. The Treatment effect was of primary interest and a significant test outcome indicates that the number of caddisflies caught in traps differed between channel units.

To test for variations in abundance at larger scales, we used ANCOVA to test for an association between numbers of females in adjacent pool–riffle pairs (i.e. Blocks in the ANOVA). In these ANCOVA, the dependent variable was numbers trapped at pools, the covariate was numbers trapped at riffles, and Time was a categorical, independent variable. The covariate effect was of primary interest and a significant test outcome would indicate a correlation between the numbers of caddisflies caught in pools and adjacent riffles, i.e. spatial autocorrelation. Where the covariate was significant, we hypothesized particular values for the coefficients (slope and intercept) describing the association between abundances in riffle–pool pairs (Table 1, Patterns A and C) and these were tested using t-tests. Thus for Pattern A, the slope of the line should be 1 and the line should intersect the origin, because a lack of response to types of channel unit means that adjacent riffle and pool samples are simply sub-samples of the number of females in that part of the channel (i.e. samples from adjacent riffle–pool pairs estimate the same thing). In contrast, for Pattern C the slope should differ from 1, or the y-intercept should differ from 0, depending on whether females associate more with pools or riffles. Data were Log(X+1) transformed before ANOVA and ANCOVA to satisfy assumptions about heterogeneity of variances.

For wing morphology, preliminary tests identified whether metrics were correlated and, if necessary, subsequent tests were restricted to metrics that were not correlated. First, we used two-way MANOVA to test for differences in wing morphology between species that differed in abundance only between adjacent channel units vs at larger scales (Patterns A and C vs Pattern B), patterns that we predicted would be related to flight capability (Table 1). Second, because abundance patterns differed between groups of species in different families (see Results) and because evolutionary history may explain systematic differences in wing morphology between insect groups (Dudley, 2000; Tercel, Veronesi & Pope, 2018; Le Roy, Debat & Llaurens, 2019), we then tested whether wing morphology differed between families. For this MANOVA, the main effect was Family, Species were nested within Families (Species were replicates), and the different wing metrics were dependent variables. If this second test revealed family-level biases in wing morphology, then the first test (testing whether wing morphology differed with scales of spatial variation) was repeated using a reduced set of metrics to avoid potential confounding effects of phylogenetic history.

3. RESULTS

Local abundance patterns for mature females of all five species of Hydrobiosidae clearly fit Pattern B (Table 1), the two species of Leptoceridae and one Hydropsychidae (Asmicridea edwardsi) fit Pattern A, one hydropsychid (Cheumatopsyche AV3) fit Pattern C most closely, and no species fit Pattern D. Preliminary

8 analyses indicted that patterns were the same in the Little and Steavenson Rivers, so the results were pooled across rivers. Males of some species were uncommon in our samples, e.g. only 16 individuals of Taschorema evansi were collected in the entire survey, in contrast to females, which were ~6´ more abundant. Teneral females (i.e. recently emerged) were present but also rare (<13% for any species) and were excluded from analyses. Most females (>80%) were clearly carrying mature eggs or had recently oviposited, and these were combined for hypothesis tests. Teneral females were excluded from analyses. Significantly more mature females were trapped at riffles than pools for six out of the nine species, including all species in the family Hydrobiosidae, and one hydropsychid (Fig. 1, Table 2). Abundances did not differ between riffles and pools for the remaining three species. There was no temporal consistency in the number of individuals trapped at locations, i.e. no particular riffle (or pool) consistently collected more females than any other. Overall female abundance varied between sample months for several species, but there were no significant Treatment ´ Time interactions. Positive associations between numbers of females trapped at adjacent pool–riffle pairs occurred in four species (Fig. 2, Table 3), indicating spatial variations in abundance over larger scales. Significant associations occurred for all species in the families Hydropsychidae and Leptoceridae, but none in the family Hydrobiosidae. In all cases of significant correlations between pool–riffle pairs (Fig 2f-i), slopes of the lines describing the relationship did not differ from 1 and y-intercepts did not differ from 0. Although Cheumatopsyche AV3 fit Pattern C most closely (significant riffle–pool differences and variations over larger scales), coefficients for the regression equation (Fig. 2f) were inconsistent with expectations (Table 1). It is possible that the slope (b = 0.69) was significantly <1 but the test lacked sufficient power to detect this statistically (slopes for the other three species were closer to 1.0, ranging between 0.92 and 1.06). In contrast, the riffle–pool differences for Cheumatopsyche AV3 were large (> 2´ more females at riffle than pools, Fig. 1). On balance, we cautiously suggest that Cheumatopsyche AV3 did fit Pattern C, with abundance varying both between types of channel unit and over larger scales.

The species set encompassed a variety of wing sizes and shapes, with ranges in female wing span and area of

6 –15 mm and 15 – 88 mm, respectively, and ranges in the dimensionless aspect ratio and �̂ (�) of 4.4 – 6.3 and 0.495 – 0.528, respectively. The values of all four metrics for each species and sex are shown in Table S1, although our analyses focussed on females only. Among the four metrics, only female wing span and wing area were correlated (r8 = 0.99, P < 0.001), so wing area was removed from subsequent analyses. Wing morphology differed significantly between species that did vs did not show spatial variation at larger scales

(Pillai's trace = 0.720, F3, 6 = 5.14, P = 0.043) and univariate tests within the MANOVA indicated that �̂ (�) was the strongest predictor of this pattern (F1, 8 = 9.98, P = 0.013) (see Table S2 for full statistical summary).

However, wing morphology differed between families (Pillai's trace = 1.26, F6, 15 = 3.41, P = 0.033) and this was due to differences in one shape variable, �̂ (�) (F2, 7 = 65.4, P < 0.001), but not wing span or aspect ratio (Table S3 for full statistical summary). Species fitting Pattern B (differences between riffles and pools only: members of the family Hydrobiosidae), had the highest values of �̂ (�). This metric can range between a

9 maximum of 0.70 and a minimum of 0.42; observed, mean values were 0.523, 0.517 and 0.490 for the families Hydrobiosidae, Hydropsychidae and Leptoceridae, respectively (Table S1). A repeat of the first test, using wing span and AR only, suggested that wing morphology did not differ significantly between species that did vs did not show spatial variation at larger scales (Pillai's trace = 0.101, F2, 7 = 0.395, P = 0.688; full summary in Table S4).

4. DISCUSSION

How adult aquatic insects are locally distributed along rivers is poorly documented, for especially sex- and age-specific distribution patterns, despite their potential value in understanding life history events that may affect population size (Peckarsky, Taylor & Caudill, 2000; Encalada & Peckarsky, 2012; Lancaster & Downes, 2014), and in elucidating consumer–resource interactions for both aquatic and terrestrial life stages (e.g. Harrison & Hildrew, 2001). The abundance of some adults perpendicular to river channels and with variations in land use or riparian vegetation has been described (Kovats, Ciborowski & Corkum, 1996; Petersen et al., 2004; Carlson et al., 2016), but descriptions of distribution patterns along channels are scarce. We identified three distinctly different and species-specific patterns in the local abundance of mature female caddisflies along short lengths of river channels with homogeneous riparian cover. Five species varied in abundance between channel units only (riffles vs pools), three varied over larger scales only (lengths encompassing multiple channel units), and one varied over both. There are no established methods for describing distribution patterns at these scales and our survey design provided clear outcomes. Our analyses focused on mature females and observed patterns that likely reflect behaviours associated with oviposition. Distribution patterns may differ for males or younger females (e.g. Petersen et al., 1999), as suggested by the paucity of males and teneral adults of some species in our survey. For some species, female aggregations clearly reflected locations where oviposition sites were locally abundant. The degree of longitudinal movement predicted to create the patterns did not correspond to wing morphology (a proxy for flight capability), but unmeasured aspects of behaviour (e.g. flight propensity) may have contributed to the observed patterns.

Oviposition sites (emergent rocks) for the five hydrobiosid species are most abundant in riffles and females were also most abundant at riffles. These variations between channel units were strong: zero hydrobiosids were collected over pools in ~20–60% of pool–riffle pairs, even when females were abundant at adjacent riffles <20 m away; and the proportion of such zeros was much lower (~8%) in other families. We are unaware of any other studies showing that local abundances of mature females can be linked so strongly to the provision of oviposition habitat. Following our hypotheses, the scarcity of hydrobiosids caught at pools coupled with the lack of association between numbers over riffles and pools along whole channel lengths, suggests that mature females of these hydrobiosids do not fly often or far along river channels, an observation consistent with reports of low movement frequencies along streams for some other caddisflies

10 (Svensson, 1974). This result was unexpected for at least one hydrobiosid, Ethochorema turbidum, because it has the biggest wings in our set of species, and hence was expected to be a strong flier (further discussion of wing morphology below). Previous studies suggest that hydrobiosids do fly long distances (Graham, Storey & Smith, 2017; Bovill, Downes & Lancaster, 2019) and such long-distance movement may reflect behaviours of young adults (Svensson, 1972; Svensson, 1974), movement at different times of day or in different locations, e.g. away from river margins, close to the ground or high in the canopy (Svensson, 1974; Collier & Smith, 1995; Didham et al., 2012).

Larger-scale variations in abundance between river lengths encompassing multiple channel units occurred in four species. Oviposition habits of three species (the two leptocerids and one hydropsychid) are not associated with riffles or pools (discussed above). In contrast, the fourth species (a hydropsychid, Cheumatopsyche AV3) lays eggs primarily on emergent rocks in riffles and, accordingly, mature females were most abundant at riffles. The absence of differences between channel units for the other hydropsychid, Asmicrdea edwardsi, which does not lay eggs preferentially on emergent rocks (Reich, 2004), lends support to our hypotheses about the role of oviposition behaviours in driving distribution patterns of mature females, even among closely related taxa. From a set of nine species, there is a probability of only P = 0.012 that the same six species known to oviposit primarily in riffles would also differ in abundance between channel units, i.e. our observations are unlikely to have occurred by chance.

Wing morphology is related to flight performance and capability of insects (Ellington, 1984a; Dudley, 2000), but we found no relationship between wing morphology and the distribution patterns of mature female caddisflies. Contrary to our hypotheses, the species with the biggest wings (Ethochorema turbidum) and theoretically a strong flier within this set of species, varied in abundance between different channel units only. Conversely, abundances of the species with the smallest wings (Cheumatopsyche AV3) varied between different channel units and over larger scales. In terms of wing shape and consistent with our hypothesis, the species with the largest aspect ratio (Notalina bifaria) varied over large scales, but so did the species with the smallest aspect ratio (Asmicridea edwardsi). Our sample size was small (nine species over three families) and a larger, more taxonomically diverse set may be required to detect associations (Tercel, Veronesi & Pope, 2018; Le Roy, Debat & Llaurens, 2019). Alternatively, wing morphology may be associated with distribution patterns and flight of younger adults or over longer distances (Lancaster & Downes, 2017), or may be associated with some other aspect of movement, such as where in the riparian zone different species fly (e.g. at canopy level or in the understorey: DeVries, Penz & Hill, 2010). It is interesting that the second moment of wing area, �̂ (�), was significantly higher for Hydrobiosidae and the associated agility and manoeuvrability (Ellington, 1984a; b) may facilitate the fine scale movements of female hydrobiosids when selecting emergent rocks for oviposition (Reich & Downes, 2003b; Reich et al., 2011; Lancaster et al., in press). This interpretation is speculative, however, and requires further investigation. Although wing morphology was not associated with distribution patterns, it is possible that some aspect of flight behaviour

11 (e.g. propensity) was associated with our hypotheses regarding the degree of longitudinal movement required to generate particular patterns

The patterns of larger-scale variations in abundance along the stream were distinct, but how they arise is less clear. Possibly, our initial assumption that mature females reach the river at random locations may be incorrect. Movements of females through the riparian zone could interact with vegetation structures and lead to aggregations in particular locations along a channel, even if the riparian vegetation appears relatively homogeneous to humans. The movement of individual insects across space can vary with species-specific behaviour and landscape features (e.g. Schultz, Franco & Crone, 2012). The lack of temporal consistency in where females were most abundant suggests that aggregations were not associated with any particular channel length or landscape feature. Temporal variability in the way movement and habitat structure interact, e.g. with weather, could mitigate against aggregations arising repeatedly in the same location, but this is speculative. Alternatively, if mature females do fly frequently along streams, local aggregations could arise by chance, analogous to the 'phantom traffic jams' or clusters of cars that can occur on roads (Kerner & Konhäuser, 1993).

The ecological functions of movement and the spatial distribution patterns that movements can create are diverse for most organisms. Freshwater ecologists, however, have typically viewed movement of adult aquatic insects in the context of dispersal between populations and examined movement away from water bodies (references above). Accordingly, there is a common assumption that flight capability (often based on measures of wing morphology) is closely associated with dispersal distances, and therefore that flight capability should explain species-specific distribution patterns over very large scales (Malmqvist, 2000; Rundle et al., 2007) or within metapopulations. While these approaches have merit, it is a rather narrow view of the diverse ecological functions of flight throughout the adult stage. Wing morphology may be more closely related to other life history events, such as mating success (Petersson, 1995) or oviposition site selection, and different elements of wing morphology may relate to sex-specific functions. Moreover, other kinds of spatial patterns generated by movement are often overlooked. In particular, flight capacity that permits long distance travel may be unrelated to movements over small scales as females search for oviposition habitat, and yet these movements determine where eggs are laid at the terrestrial–aquatic life stage transition. For some stream taxa, we know that the provision of oviposition habitat is correlated with egg mass density (Peckarsky, Taylor & Caudill, 2000; Lancaster, Downes & Arnold, 2010a; Lancaster & Downes, 2018), which can sometimes explain larval densities along channels (Encalada & Peckarsky, 2012; Lancaster & Downes, 2014). The number of egg masses at a location is a function of egg-laying success coupled with the supply of gravid females. Our data suggest that mature females have species-specific propensities for flying along channels in search of suitable egg-laying habitat. Thus, the hydrobiosids in this study may only move to the nearest riffle, whereas Cheumatopsyche AV3 may move across multiple channels units, suggesting that Cheumatopsyche AV3 has a greater capacity to respond to riffles offering

12 high densities of egg-laying habitat. More generally, a more sophisticated and diverse approach to research on flight and the spatial distribution of adult aquatic insects is required to provide a comprehensive understanding of the diverse ecological roles of flight and the links between aquatic and terrestrial life stages.

ACKNOWLEDGEMENTS This research was supported by a Discovery grant from the Australian Research Council (DP 160102262). We are indebted to Alena Glaister for help collecting, identifying and preparing specimens for measuring wing metrics, and to Ros St Clair for assistance identifying leptocerid caddisflies. Field work was carried out in conjunction with a Research Permit (No. 10007855) under the National Parks Act (Australia), from the Victorian Department of Environment, Land, Water and Planning.

DATA AVAILABILITY Data will be made available upon reasonable request to the authors.

CONFLICT OF INTEREST To the authors' knowledge, there are no conflicts of interest.

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17

Figure 1 Abundance variations between types of channel unit: Number of adults (mean ± SE) trapped at pools and riffles over three months, for nine species of caddisfly: (a)–(e) family Hydrobiosidae, (f) & (g) family Hydropsychidae, (h) & (i) family Leptoceridae. Asterisks in panels (a)–(f) indicate a significant treatment effects, i.e. differences between riffles and pools. See Table 2 for summary of statistical analyses.

18

Figure 2 Abundance variations over larger scales: Number of female adults trapped at pools in relation to the number at adjacent riffles over all sample times, for nine species of caddisfly: (a)–(e) family Hydrobiosidae, (f) & (g) family Hydropsychidae, (h) & (i) family Leptoceridae. Riffle–pool pairs with zero adults in both locations were omitted from analyses. Solid lines in panels (f)–(i) indicate a significant relationship between numbers in pools and riffles; dashed lines indicate 1:1 relationships, i.e. a line with a slope of 1 and intercept of 0. See Table 3 for summary of ANCOVA; panels (f)–(i) report results of t-tests to determine whether the line slope b ≠ 1 and the intercept a ≠ 0 (see Table 1).

19 Table 1. Four possible combinations (Patterns A–D) of outcomes for two statistical tests that determine whether female abundance differs between different channel units (riffles vs pools) and/or at larger scales (channel lengths encompassing multiple channel units). Interpretation of how the patterns arise is based on whether oviposition sites are associated with riffles or pools, and the expected degree of longitudinal flight required to generate the pattern. The degree of longitudinal flight may correspond to flight capability, as measured by wing morphology. Patterns A and C both involve larger scale variations in abundance, but in the ANCOVA we expect different values for coefficients of the line describing the relationship between numbers in pools and in adjacent riffles: b = line slope, a = intercept. See Section 2.4 Statistical analyses for more detailed discussion of statistical tests and inferences.

Test Riffle–pool Larger scale Flight capability Pattern difference variations Interpretation or propensity (ANOVA) (ANCOVA) A NS Significant Location of oviposition sites unrelated to Strong b = 1 and a = 0 channel units; longitudinal movement creates larger scale spatial variation B Significant NS Oviposition sites most abundant in one type Weak of channel unit; little longitudinal movement C Significant Significant Oviposition sites most abundant in one type Strong b ≠ 1 or a ≠ 0 of channel unit AND longitudinal movement creates larger scale spatial variation (e.g. some riffles are more attractive than others) D NS NS Location of oviposition sites unrelated to Weak channel units; little longitudinal movement

20 Table 2 Summary of split-plot ANOVA testing for differences in abundance between channel units: riffles vs pools. Left hand column: species of Hydrobiosidae; right hand column: species of Hydropsychidae and Leptoceridae. P-values significant at a < 0.05 are shown in bold. See Fig. 1 for illustration.

Source d.f. MS F P d.f. MS F P

Between Apsilochorema spp. Cheumatopsyche AV3 Time 2 0.0128 0.643 0.540 1 0.121 0.259 0.621 Block(Time) 15 0.0199 11 0.466 Within Treatment 1 0.0935 7.55 0.015 1 0.866 12.69 0.005 Treat. ´ Time 2 0.0048 0.391 0.683 1 0.0176 0.258 0.622 Error 15 0.0124 11 0.0682

Between Ethochorema turbidum Asmicridea edwardsi Time 2 0.0371 1.12 0.351 2 4.22 48.5 <0.001 Block(Time) 15 0.0331 15 0.0870 Within Treatment 1 0.167 5.76 0.030 1 0.0306 1.14 0.303 Treat. ´ Time 2 0.0073 0.251 0.782 2 0.0009 0.035 0.966 Error 15 0.0290 15 0.0269

Between Taschorema evansi Notalina bifaria Time 2 0.0714 2.24 0.141 2 0.4499 1.77 0.203 Block(Time) 15 0.0320 15 0.2536 Within Treatment 1 0.197 12.6 0.003 1 0.0042 0.153 0.701 Treat. ´ Time 2 0.0066 0.420 0.665 2 0.0311 1.12 0.351 Error 15 0.0157 15 0.0277

Between Ulmerochorema seona Triplectides proximus Time 2 0.0767 1.288 0.305 2 2.11 15.6 <0.001 Block(Time) 15 0.0596 15 0.136 Within Treatment 1 0.1222 5.084 0.040 1 0.0659 2.75 0.118 Treat. ´ Time 2 0.0043 0.177 0.839 2 0.0064 0.267 0.770 Error 15 0.0240 15 0.0240

Between Ulmerochorema rubiconum Time 2 0.0101 1.279 0.307 Block(Time) 15 0.0079 Within Treatment 1 0.0629 7.011 0.018 Treat. ´ Time 2 0.0204 2.280 0.137 Error 15 0.0090

21 Table 3 Summary of ANCOVA testing for larger scale spatial variation in abundance, i.e. over channel lengths encompassing multiple channel units. Left hand column: species of Hydrobiosidae; right hand column: species of Hydropsychidae and Leptoceridae. Table 1 outlines expected values for coefficients (b, slope, and a, intercept) of the line describing numbers in pools as a function of numbers in adjacent riffles; t-tests of whether b = 1 and a = 0 are shown in Fig. 2. Interaction terms were not significant in any test and were removed from the model. Cases with 0 animals in both riffle and pool samples were omitted, leading to variations in the degrees of freedom between species. P-values significant at a < 0.05 are shown in bold.

Source d.f. MS F P d.f. MS F P

Apsilochorema spp. Cheumatopsyche AV3 Nos in Riffles 1 0.0003 0.022 0.886 1 1.52 12.6 0.005 Time 2 0.0016 0.118 0.889 1 0.0544 0.450 0.518 Error 11 0.0135 10 0.121

Ethochorema turbidum Asmicridea edwardsi Nos in Riffles 1 0.0098 0.478 0.503 1 0.415 7.66 0.015 Time 2 0.0180 0.873 0.443 2 0.120 2.22 0.145 Error 12 0.0206 13 0.0541

Taschorema evansi Notalina bifaria Nos in Riffles 1 0.0109 0.460 0.509 1 1.41 23.4 <0.001 Time 2 0.0472 1.992 0.176 2 0.0767 1.27 0.314 Error 13 0.0237 14 0.0604

Ulmerochorema seona Triplectides proximus Nos in Riffles 1 0.140 2.93 0.111 1 0.849 16.5 0.001 Time 2 0.0205 0.430 0.660 1 0.0075 0.145 0.866 Error 13 0.0478 10 0.0514

Ulmerochorema rubiconum Nos in Riffles 1 0.0000 0.001 0.980 Time 2 0.0056 0.713 0.510 Error 14 0.0079

22 Supplementary material

Jill Lancaster, Barbara J. Downes and Georgia K. Dwyer Terrestrial–aquatic transitions: local abundances and movements of mature female caddisflies are related to oviposition habits but not flight capability. Freshwater Biology 2020 DOI: 10.1111/fwb.13472

Table S1. Summary of wing metrics (mean ± SE) describing wing morphology of female and males of ten species of caddisfly. N = 10 for all species/sex combination, except A. gisbum males where N = 2.

2 Species Sex Span (mm) Area (mm ) Aspect ratio �̂ (�) Apsilochorema gisbum F 8.7 ± 0.18 31.7 ± 1.3 4.85 ± 0.036 0.5246 ± 0.0006 M 6.8 ± 0.95 18.5 ± 4.3 5.00 ± 0.22 0.5199 ± 0.0007 Apsilochorema obliquum F 8.7 ± 0.20 33.1 ± 1.4 4.63 ± 0.019 0.5241 ± 0.0011 M 7.9 ± 0.20 27.1 ± 1.4 4.65 ± 0.054 0.5233 ± 0.0011 Ethochorema turbidum F 14.9 ± 0.22 87.6 ± 2.6 5.10 ± 0.035 0.5218 ± 0.0010 M 12.1 ± 0.19 58.9 ± 1.7 4.95 ± 0.053 0.5258 ± 0.0010 Taschorema evansi F 9.2 ± 0.18 34.1 ± 1.3 5.00 ± 0.029 0.5233 ± 0.0011 M 7.2 ± 0.16 20.4 ± 1.0 5.17 ± 0.053 0.5220 ± 0.0010 Ulmerochorema seona F 7.0 ± 0.20 20.5 ± 1.1 4.77 ± 0.035 0.5281 ± 0.0009 M 6.2 ± 0.15 16.5 ± 0.9 4.72 ± 0.049 0.5287 ± 0.0010 Ulmerochorema rubiconum F 8.4 ± 0.28 26.2 ± 1.8 5.14 ± 0.085 0.5207 ± 0.0018 M 7.2 ± 0.17 19.7 ± 1.0 5.31 ± 0.044 0.5192 ± 0.0007 Cheumatopsyche AV3 F 6.3 ± 0.05 15.3 ± 0.30 5.20 ± 0.030 0.5172 ± 0.0014 M 6.4 ± 0.06 15.7 ± 0.30 5.29 ± 0.048 0.5124 ± 0.0014 Asmicridea edwardsi F 7.9 ± 0.25 28.5 ± 1.6 4.37 ± 0.095 0.5194 ± 0.0020 M 6.7 ± 0.07 24.4 ± 0.5 3.68 ± 0.025 0.5201 ± 0.0013 Notalina bifaria F 7.8 ± 0.08 19.7 ± 0.33 6.25 ± 0.078 0.5008 ± 0.0009 M 6.4 ± 0.06 37.4 ± 0.96 6.06 ± 0.056 0.4837 ± 0.0007 Triplectides proximus F 12.5 ± 0.18 63.7 ± 1.37 4.91 ± 0.043 0.4946 ± 0.0009 M 13.7 ±0.16 69.9 ± 1.59 5.41 ± 0.036 0.4825 ± 0.0009

23 Table S2. Summary of two-way MANOVA testing whether wing morphology of females differed between species that showed spatial variation at large scales vs the smaller scale of channel units only. Univariate F-tests within the MANOVA indicate which of the wing metrics (wing span, aspect ratio and

�̂ (�)) contributed to any over-all differences.

Source df Pillai's MS F P trace Multivariate test Scale of spatial pattern 3, 6 0.720 5.14 0.043 Univariate tests Wing span Scale of spatial pattern 1 1.84 0.242 0.636 Error 8 7.59 Aspect ratio Scale of spatial pattern 1 0.170 0.653 0.442 Error 8 0.260

�̂ (�) Scale of spatial pattern 1 0.00060 9.98 0.013 Error 8 0.00006

24 Table S3. Summary of two-way MANOVA testing for differences between families with respect to three wing metrics for females: wing span, aspect ratio and �̂ (�).

Source df Pillai's MS F P trace Multivariate test Family 6, 15 1.26 3.41 0.033 Univariate tests Wing span Family 2 5.57 0.758 0.503 Species 7 7.34 Aspect ratio Family 2 0.401 1.94 0.214 Species 7 0.207

�̂ (�) Family 2 0.00051 65.4 <0.001 Species 7 0.000008

25 Table S4. Summary of two-way MANOVA testing whether wing morphology of females differed between species that showed spatial variation at large scales vs the smaller scale of channel units only. Only wing metrics that did not differ systematically between families were used: wing span, aspect ratio (Table S3). Univariate F-tests within the MANOVA indicate which metrics contributed to any over-all differences.

Source df Pillai's MS F P trace Multivariate test Scale of spatial pattern 2, 7 0.101 0.395 0.688 Univariate tests Wing span Scale of spatial pattern 1 1.84 0.242 0.636 Error 8 7.59 Aspect ratio Scale of spatial pattern 1 0.170 0.652 0.442 Error 8 0.260

26

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Lancaster, J; Downes, BJ; Dwyer, GK

Title: Terrestrial-aquatic transitions: Local abundances and movements of mature female caddisflies are related to oviposition habits but not flight capability

Date: 2020-01-09

Citation: Lancaster, J., Downes, B. J. & Dwyer, G. K. (2020). Terrestrial-aquatic transitions: Local abundances and movements of mature female caddisflies are related to oviposition habits but not flight capability. Freshwater Biology, 65 (5), https://doi.org/10.1111/fwb.13472.

Persistent Link: http://hdl.handle.net/11343/234115

File Description: Accepted version