Evidence that freshwater mussels attempt temporal partitioning of their host fishes

Abstract

Freshwater unionid mussels, which have parasitic glochidia larvae, often occur in multispecies mussel beds where they likely compete for host fishes. The temporal dynamics of glochidia release was examined at 2-h intervals over ten, 24-h periods in the Sydenham River, Ontario, Canada from late August through September 2020 using a rosette water sampler. A total of 6104 glochidia from 17 species were identified morphometrically and these were dominated numerically by four species (Eurynia dilatata; Ortmanniana ligamentina; Epioblasma triquetra; and Cyclonaias tuberculata). The total glochidia abundance was greatest during the night, 20:00 h (± 1 h) local solar time (LST) in part because of the dominant species. Conversely, the abundance of glochidia from individual species was not uniform temporally among days (p < 0.001) nor time-of-day (p < 0.001) but appeared to coincide with the reported diel activity periods (i.e., diurnal, crepuscular, and nocturnal) of the host fishes for 10 species. The abundance of glochidia peaked at different times in unionid species that shared hosts (five cases), suggesting an attempt to partition host fishes temporally. Whereas temporal partitioning of hosts could ultimately provide a mechanism by which multiple species of unionids coexist in the same habitat, this is remarkable because glochidia release occurs over longer temporal scales (i.e., weeks), and there is limited gill-space on host fish, especially small individuals that may not have acquired immunity. Recognizing the temporal dynamics of glochidial abundance in multispecies mussel beds will inform conservation approaches that address mussel-host fish relationships in this imperiled taxon.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-17584-z.

Introduction

Interspecific competition is a key factor that affects the diversity of communities^1–4. Indeed, coexisting species should not compete for the same limited resource^5–7 unless they have mechanisms to partition those resources in time or in space⁸, increase the complexity/utilization of resource space⁹, or coexist due to other factors¹⁰. Spatial partitioning occurs through the segregation of microhabitats^11–14 or resources via character displacement^15,16, whereas temporal partitioning involves accessing abiotic resources (e.g., nutrients, water use^17,18) or biotic agents (e.g., pollinators, hosts^19,20) at different times. Temporal partitioning often occurs over large scales (e.g., seasonal), is less well studied than spatial partitioning, and may be more likely in less conspicuous taxa that are tightly coupled to a finite resource pool (e.g., insect parasites²⁰).

Native freshwater mussels (Unionidae) provide an excellent model system in which to examine temporal resource partitioning because they are often found in multispecies (e.g., > 20 species) mussel beds in rivers^21,22. As suspension feeders, they are important constituents of aquatic systems^23–25 yet are one of the most endangered taxa due to habitat loss, invasive species, and commercial exploitation^26–28. Unionids compete for seston (suspended matter) with invasive bivalves^29,30 and feed selectively on different suites of algae and thus partition algal resources in multispecies beds³¹. Another source of competition relates to their reliance on host fishes for reproduction^22,32,33. A variety of host attraction strategies (e.g., fish-like lures) have evolved because successful infection of the host (encystment of parasitic larvae, glochidia) and subsequent metamorphosis and excystment of juvenile mussels that settle to the riverbed, is needed for recruitment³². There can be overlap in host use²⁵ with estimates that 68% of host fish species may be shared by two or more unionid species in some rivers³³. Importantly, host availability may be limited due to low abundance, especially high-quality primary hosts on which juvenile metamorphosis is high, limited space on the host’s gills, and/or acquired immunity of the host from previous infection^22,26,34,35. Finding fewer than 10 individuals of a given host may be common in nature, however, host limitation has not been tested under these conditions²². How and if mussels have evolved to deal with the potential for competition for host fishes remains to be addressed.

Potential segregation of host use may be possible through different host attraction strategies^32,36 or due to the different developmental strategies used by unionids^37–39. Specifically, tachytictic species typically spawn in the spring, brood larvae for short periods (e.g., few weeks), and release glochidia in the summer, whereas bradytictic species typically spawn in late summer, brood larvae for long periods (e.g., seven months) and release glochidia in early spring^38,40. The timing of spawning, brooding, and release of glochidia can vary among species, populations and geographic localities^32,38, as can host-fish use within a given species^33,41. Temporal partitioning has been suggested for two congeneric species that display lures in the day versus night⁴². Unfortunately, most of these observations are based on associations and laboratory results rather than through field observations, especially of glochidia presence in the seston over short-time scales pertinent to the diel activity patterns of their host fish⁴³.

We hypothesize that unionids may partition their hosts through the release of glochidia at different temporal scales related to the diel activity patterns of their host fish species, which have different temporal patterns in their locomotion, feeding and spawning activities⁴⁴. Diel is defined as a 24-h cyclic pattern based on light–dark cycles: diurnal (daytime); crepuscular (twilight periods, dawn and dusk); and nocturnal (nighttime). We predict that the abundance of glochidia from different species will vary on a diel basis and in relation to the reported diel activity patterns of host fishes, especially in species that share the same host fishes. We examine this prediction by sampling the water column in a multispecies mussel bed over ten, 24-h periods at a frequency of 2 h (i.e., fine temporal scale within a short period) using a custom rosette sampler and identifying the glochidia using shape and morphometric features.

Methods

Glochidia sampling

A custom-built rosette sampler was used to collect glochidia in the Sydenham River at Florence, Ontario, Canada (42.65007, −82.00903; mussel bed size ~ 20 × 20 m), which has a high species richness (24 species; 4.6 ± 0.1 species m⁻²) and mussel density (23.6 ± 2.3 mussels m^{−2 45}). Sampling in the spring and early summer was not possible due to the onset of the COVID-19 pandemic; sampling took place between August 31 and September 28, 2020. A total of 20 species are tachytictic brooders and/or are in a gravid state with the potential to release glochidia during the sampling period (i.e., 12 observed by the Ackerman Laboratory or Fisheries and Oceans Canada [DFO]; and/or 8 reported in³⁸ and/or⁴⁶; Table S1). Backpack electrofishing and seine netting data of the abundance of fish species caught at Florence during August and September 2020, and abundances since 2009 were obtained from⁴⁷.

Glochidia were sampled continuously over ten, 24-h periods using a submersible pump (240 L/h submersible pump; DC30A-1230 Brushless 12 VDC Pump, Shysky Tech, Guangdong, China) that delivered water from 14 cm above the riverbed. The pump was held in position on steel rebar ensuring that the 6 mm ID (inner diameter) intake faced into the flow; the pump was placed in a 5 mm polypropylene mesh bag to prevent clogging. River water flowed into a turntable containing 12 PVC sample chambers (100 μm Nitex mesh bottom), which rotated every 2 h (i.e., 12 × 2-h samples in 24 h), and the water returned to the river via an overflow tube; chamber positioning and turntable rotation was verified before each sampling day; see Figure S1). Sampling was generally started in the late morning to early afternoon, i.e., 11:59 ± 0:32 (mean ± SE; n = 10) h local solar time (LST), which was used to account for the position of the sun in the sky at the sample location (i.e., solar noon occurs when the sun reaches its highest position in the sky⁴⁹). This enables the comparison of similar diel periods over the four-week sampling period when seasonal changes were occurring.

We acknowledge that the Nitex mesh may not retain glochidia with dimensions < 100 μm, e.g., Potamilus fragilis and Quadrula quadrula⁴⁸, but it allowed sufficient flow to ensure that overflow from the chamber did not occur due to blockage. The volumetric flow rate of the system at the overflow tube was measured at 0 h and at 24 h of a 24-h sampling period to identify variations in the pump flow, which may have been caused by the accumulation of small particles in the pump. The pump and mesh was cleaned at 24 h to ensure that it was clean on the next deployment (Fig. S1).

Glochidia identification

Samples were imaged with a 10-megapixel camera (Swiftcam Sc1003-CK, Swift Optical Instruments, Inc., Schertz, TX, USA) attached to a stereomicroscope (Nikon SMZ-2 T; Tokyo, Japan). Each glochidium (bivalve or detached valve) was assigned to shape category: semi-circular; elliptical; triangular; or axe-shaped (unique to Potamilus alatus, Pink Heelsplitter, in the river; Fig. 1A). The shell length (SL), hinge length (HL) and shell height (SH)⁴⁸ were measured to the nearest 0.01 μm in Swift Imaging (ver. 3.0) (Fig. 1B). A reference collection of glochidia obtained from gravid female mussels in the region (i.e., Glochidia Atlas of Ontario [Ackerman Laboratory, unpublished, University of Guelph]⁵⁰) was used to identify glochidia. Species included extant species at the Florence site⁴⁵ with glochidia shell dimensions > 100 μm that have also (1) been observed to be gravid in southern Ontario or North America where local data is missing and/or (2) potentially releasing glochidia during late August and September (Table S1). Three sets of biplots (i.e., SH vs. SL, SH vs. HL, and SL vs. HL) were made for each shape (excluding axe-shape) with species dimensions (mean ± 2 SD) from the reference collection (Figure S2).

Fig. 1.

Shape and morphometry of imaged unionid glochidia examined: (a) comparison of shape categorizations for (A) semi-circular, (B) elliptical, (C) triangular, and (D) axe-shaped glochidia; and (b) shell height, hinge length, and shell length of a glochidium.

Species identification models were created in Microsoft Excel using a series of nested logical statements, and separate models were applied to semi-circular, elliptical and triangular glochidia. Each model queried whether the SH, HL, and SL were within the mean ± 2 SD of the reference collection, and all three metrics were used for species identification. The order of species within a nested model was sensitive for species with overlapping measurements, which affected the numerical dominance of the taxon identified. Therefore, in the model used (of eight; Table S2), priority was given to species with gravidity periods and reported release periods at the time of sampling and was based on species numerical density at the site⁴⁵. The greatest overlap was among semi-circular glochidia (Epioblasma rangiana, Northern Riffleshell; Epioblasma triquetra, Snuffbox; and Eurynia dilatata, Spike). The model was verified by comparing the species identified to the biplot morphometric graphs (Figure S2).

After identification, individual valves within a given species were paired if they were within ± 4 μm for each measurement (SH, HL, and SL) to provide a measure of the number of glochidia collected. The number of glochidia collected in each chamber was then divided by the volume of water sampled (m³) in the 2-h period (adjusted for any differences observed between 0 and 24 h, assumed to be linear), which provided a concentration of glochidia (glochidia m⁻³). The observed glochidia concentration (glochidia m⁻³) was multiplied by a constant volume (0.283 m³, which was the largest volume of water sampled in 2 h) for each 2-h interval. This provided the abundance of glochidia in each 2-h interval required for the statistical analyses.

Statistical analyses

A generalized linear mixed model (GLMM) was used to determine whether the abundance of glochidia was equal at different times. Species that represented > 2% of the total number of glochidia collected were used because of the difficulties of applying statistical analysis associated with large numbers of zero observations⁵¹. Species and time-of-day (i.e., individual 2-h sample chambers) were treated as fixed effects, and sampling day was a random effect. A negative binomial distribution with a log-link was used to account for overdispersion. Pairwise comparisons based on the least-square (LS) means were examined with Tukey–Kramer adjustments when significant factors were determined. A covariance parameter test was run to determine if the variance component of the random effect was equal to zero.

Chi-square () goodness-of-fit tests were used to determine whether there were temporal patterns in the distribution of glochidia sampled, based on (1) the time-of-day and (2) the sampling day. Three probability distribution models were examined, including: (i) uniform through time; (ii) random in time using a Poisson distribution; and (iii) clustered in time using a negative binomial distribution⁵². When those models failed, a analysis of frequency was used to determine whether the temporal distribution of glochidia followed diurnal, crepuscular, or nocturnal diel periods. Separate analysis of frequency tests were also run to examine the sensitivity of the glochidia identification model, specifically by comparing the model used in the study to the two models that were most different from it (Table S3).

Results

A total of 5245 detached valves and bivalves (i.e., intact glochidia), which were collected during the 10 sampling days, were identified to species, except for 217 valves that were broken and/or unidentifiable. The separation and breakage of valves may have occurred during sample collection from the Nitex mesh in the field and/or processing in the laboratory in which glochidia were separated from detritus. After pairing valves within species, a minimum of 3568 glochidia identifications was obtained (55% were single valves). This number was adjusted to 6104 glochidia after corrections for variations in volumetric flow rates among days were made. Total glochidia abundances varied both within and among days (508.7 ± 31.2 [mean ± SE] glochidia per 2-h interval, and 610.4 ± 99.5 glochidia daily; Figure S3 and S4), but the abundance of glochidia declined over the four-week sampling period. The abundance of glochidia increased during the dusk crepuscular period, peaked during the nocturnal activity period (for 8 of 10 days) and declined by the following diurnal period. Peak abundances were observed during a diurnal and a crepuscular activity period on the other two sampling days, respectively.

Seventeen unionid species were identified; E. dilatata was the most abundant species (40.1 ± 2.7 glochidia) in a given 2-h sample period, whereas Paetulunio fabalis (Rayed Bean) was the least abundant (0.01 ± 0.01; Figure S5). Statistical analysis focused on four main species that individually contributed > 2% of the total abundance of glochidia (E. dilatata = 74.08% of the total glochidia collected; O. ligamentina = 9.20%; E. triquetra = 2.87%; and C. tuberculata = 2.01%). The remaining thirteen species had low glochidia abundances with averages of less than one glochidium per 2-h sample (Figure S5). Glochidia abundance for the four main species differed in abundance in the 2-h samples (F_33,414 = 556.11, p < 0.001) and all pairwise differences were significant (Tukey–Kramer adjusted LS means p < 0.05). There was a significant effect of time-of-day (i.e., 2-h periods) for the main species pooled among the ten sampling days (F_11,414 = 3.20, p < 0.001). The average number of glochidia was significantly higher at 20:00 h (± 1 h LST), corresponding to crepuscular into nocturnal diel periods and pairwise differences were detected between 20:00 h and diurnal periods (10:00–18:00 h, LS means p < 0.03; Fig. 2). Each of the main species had relatively high abundances at 20:00 h, which corresponded to the peak abundance for E. dilatata and O. ligamentina. The highest abundance was at 06:00 h for C. tuberculata and E. triquetra corresponding to crepuscular into diurnal diel periods (Fig. 3). Glochidia abundance was highest at different times of the day for different species, i.e., there was a significant interaction of species and time-of-day (F_33,414 = 1.59, p < 0.0228). The abundance of E. dilatata was significantly greater than the other main species regardless of the time-of-day based on Tukey–Kramer multiple comparisons (p < 0.001).

Fig. 2.

Average (± 1 standard error) abundance of glochidia pooled among main species (i.e., Cyclonaias tuberculata, Epioblasma triquetra, Eurynia dilatata, and Ortmanniana ligamentina) over a 24-h period. Bars with the same letter are not significantly different (α = 0.05) based on the Tukey–Kramer LS means. The horizontal bar indicates the diel period (blue = Diurnal, yellow = Crepuscular, and pink = Nocturnal).

Fig. 3.

Average (± 1 standard error) abundance of glochidia from main species (i.e., Cyclonaias tuberculata, Epioblasma triquetra, Eurynia dilatata, and Ortmanniana ligamentina) during 2-h intervals for each sampling day. The horizontal bar indicates the diel period (blue = Diurnal, yellow = Crepuscular, and pink = Nocturnal).

Diel pattern in glochidia abundance

The distribution of glochidia from all species pooled was not uniform among days (, p < 0.001) nor time-of-day (, p < 0.001), and did not satisfy a random or a clustered distribution among days (, p < 0.001, and , p < 0.001, respectively) nor time-of-day (, , p < 0.001). Similar results were obtained for the individual main species (i.e., neither uniform, nor randomly distributed nor clustered among days or time-of-day; p < 0.001). The analysis of frequency of the temporal distribution of glochidia abundance revealed, however, significantly higher abundance of glochidia during certain diel activity periods (i.e., seven nocturnal, one diurnal) in eight species of the 17 species (one of the eight species had limited data and violated the assumptions of , i.e., expected < 5; Table 1). We note that these outcomes did not differ substantially using different glochidia identification models (Supplemental: Species Identification Model Comparison; Table S2 and S3).

Table 1.

goodness-of-fit analysis of glochidia occurrence in the water column during each diel period.

Species	Relative deviation				P value
	Diurnal	Crepuscular	Nocturnal
Cyclonaias tuberculata	0.199	− 0.471	− 0.120	7.01	0.030
Epioblasma rangiana	− 0.283	− 0.074	0.458	6.41	0.040
Epioblasma triquetra	− 0.012	− 0.136	0.069	0.76	0.685
Eurynia dilatata	− 0.130	− 0.085	0.229	131.06	< 0.001
Fusconaia flava	− 0.351	0.019	0.524	17.32	< 0.001
Lampsilis cardium	− 0.092	− 0.074	0.166	0.58	0.746
Lasmigona costata	− 0.009	− 0.451	0.188	2.14	0.343
Ortmanniana ligamentina	− 0.257	− 0.188	0.463	65.82	< 0.001
Pleurobema sintoxia	− 0.442	0.235	0.579	10.79	0.004
Potamilus alatus	− 0.212	0.017	0.315	2.94	0.230
Ptychobranchus fasciolaris	− 0.388	0.087	0.555	13.91	0.001
Cyclonaias pustolosa*	− 1	0.235	1.429	7.39	0.025
Lasmigona complanata*	0.071	− 0.407	0.049	0.65	0.724
Ligumia recta*	− 0.180	0.764	− 0.028	2.02	0.365
Paetulunio fabalis*	0.912	− 1	− 1	1.83	0.401
Pyganodon grandis*	0.043	− 0.327	0.060	0.37	0.833
Strophitus undulatus*	0.912	− 1	− 1	1.83	0.401

Positive relative deviations (i.e., [observed frequency–hypothesized frequency] / hypothesized frequency) indicate a higher incidence than expected (i.e., proportional to the length of each diel period within a day). Significantly higher abundances are presented in bold font. * = species with limited data and expected abundances < 5.

Mussel-fish interactions

Seventeen mussel species and 61 potential host fish were surveyed in the Sydenham River leading to high numbers of potential interactions for unionid mussels that are considered generalist species in terms of host use (e.g., 27 fish species for Strophitus undulatus [Creeper]; 22 fish species for Pyganodon grandis [Giant Floater]; Figure S6). Conversely, the diel period(s) in which a given species considered a specialist had its higher abundance of glochidia in the water column typically aligned with the reported diel activity pattern of one or more of its host fishes. This was the case for 10 out of 11 unionid species that were observed over all three diel periods with sufficient abundance (i.e., ≥ 5 glochidia per diel period to allow for a test; Table 1). For example, the highest abundance of P. alatus was during the nocturnal and crepuscular periods, which corresponds to its nocturnally active host fish Aplodinotus grunniens (Freshwater Drum). One exception is the host-parasite relationship for diurnally abundant C. tuberculata and their nocturnal catfish hosts.

Unionid specialists that share ≥ 2 hosts exhibited temporal differences in glochidia abundance (Figure S6, S7, and S8; Table 2). The peak abundances of glochidia differed among days and/or times of the day for 9 of the 11 host fish species shared by more than one unionid (Table 2). The 11 host fishes were reduced to 5 because 3 host fish species were not observed and too few glochidia (i.e., < 40 glochidia in total) were observed to include them (3 fish species) in the analysis. Regardless, differences in timing (i.e., allochrony) of glochidia abundance was observed among unionid species that shared these host fishes (Table 2). For example, Etheostoma nigrum is used as a host by two unionid species that have a peak abundance at different times (E. rangiana at 20:00 h and P. fasciolaris at 00:00 h). This is also the case for P. caprodes, which is used as a host for E. rangiana and E. triquetra (peaks at 20:00 h and 06:00 h, respectively). The situation for Perca flavescens (Yellow Perch) is even more surprising because it is used by three unionids that have a peak abundance in glochidia at different times (E. dilitata at 20:00 h, L. cardium at 06:00 h, and O. ligamentina at 22:00 h). Similar patterns of temporal potential separation of hosts were observed among unionids for all 5 host fishes (Table 2).

Table 2.

Comparison of peak abundance in glochidia among days and times of the day for unionid specialists that share host fishes.

		Specialist mussel species
		Cyclonaias tuberculata	Epioblasma rangiana	Epioblasma triquetra	Eurynia dilatata	Lampsilis cardium	Ortmanniana ligamentina	Ptychobranchus fasciolaris	Cyclonaias pustolosa	Paetulunio fabalis
Peak abundance among days		Aug. 31–Sept. 17	Aug. 31–Sept. 16, Sept. 23–24	Aug. 31–Sept. 17	Aug. 31–Sept. 17	Aug. 31–Sept. 17	Sept. 11–16	Aug. 31–Sept. 28	Sept. 27–28, Sept. 16–20	Limited data
Peak abundance within a day (first and second highest)		06:00 and 20:00	22:00 and 20:00	06:00 and 20:00	20:00 and 22:00	06:00 and 00:00	22:00 and 20:00	00:00 and 04:00	Limited data	Limited data
Fish abundance at Florence (2020, 2009–2020 average ± SE, n = number of surveys)47	Ameiurus melas (0, N/A, n = 1)	X							X
	Culaea inconstans (N/A, 1, n = 1)		X	X				X
	Etheostoma nigrum* (16, 22 ± 5; n = 23)		X					X
	Ictalurus punctatus (2, 1.1 ± 0.4, n = 18)	X							X
	Lepomis cyanellus* (0, 1 ± 0; n = 2)					X	X
	Lepomis macrochirus (0, 1, n = 1)					X				X
	Micropterus salmoides* (3, 2.6 ± 0.4, n = 10)					X	X
	Perca flavescens* (0, 1, n = 1)				X	X	X
	Percina caprodes* (13, 9.4 ± 1.9; n = 22)		X	X						X
	Pomoxis annularis (N/A, N/A)				X	X	X
	Pomoxis nigromaculatus (0, N/A)				X		X

Legend: X = potential unionid-host fish relationships, and fish species with multiple X indicate overlap in potential host use; * = fish present in the river that overlap with two or more unionids with abundant glochidia, which are indicated by grey highlight. Fish abundance at the Florence site (42.65007, −82.00903) in 2020 and the average ± SE in August and September for multi-seasonal surveys (n) between 2009 and 2020⁴⁷ are listed in parenthesis; “N/A” indicates that the species was not included in the surveys.

Discussion

The results provide support for the hypothesis that different co-occurring freshwater mussel species have specialized niches⁵³ through the release of glochidia at different times to minimize potential competition for host fishes. This would be analogous to the temporal partitioning that occurs at seasonal scale (e.g., tachytictic species release glochidia in summer vs. bradytictic species which release in spring³²), but on diel scale. The occurrence of glochidia in the water column was not uniform, random, nor clustered on short-time scales, rather, significant temporal patterns in glochidia abundance were found among diel periods (i.e., diurnal, crepuscular, and nocturnal), which coincided with the reported diel activity pattern of one or more of their host fishes (i.e., higher glochidia abundance occurred during host diel activity periods in 10 out of 11 unionid species). Moreover, and remarkedly, when a specific host fish is shared by more than one unionid in the community, the timing in the peak of glochidia abundance differed among the unionid species, as was observed among five fish species in the Sydenham River. These results are consistent with the predictions²⁰, namely that temporal portioning is likely more common in taxa that are tightly coupled to their finite resource pool (e.g., host fishes). Whereas mussel species appear to specialize on infesting their host fishes at different times that align with the time that their hosts are most active, there are factors beyond a mussel species’ control that limit the success of temporal partitioning. Specifically, fish are active at different times and are constantly moving⁴³, they can be infested by different unionid species⁵⁴, they exhibit acquired immunity^21,22, and space for glochidia and/or host availability may be limited in the wild²², especially small individuals that may not have acquired immunity. Consequently, the unionid attempts at temporal partitioning are not likely to become fixed.

Analysis of peak glochidia abundance among mussel specialists that share hosts revealed that there are temporal differences within and among days, which is consistent with competition among unionids for host fishes. Previous studies indicate that the abundance of some fish species may be low in the Sydenham River⁴⁷, which would also drive the intensity of competition⁵³. For example, P. caprodes is a crepuscularly active fish used by two Epioblasma species^33,55, one at dawn vs. the other at dusk (E. triquetra peak abundance at 06:00 h, and E. rangiana peak abundance at 20:00 h; there was limited data for P. fabalis and S. undulatus, which are also reported to use P. caprodes³³). This is similar to two Villosa species with different lure coloration and diel rhythms, which target host fish at opposing times (i.e., diurnal and nocturnal periods⁴²). Ortmanniana ligamentina (peak abundances at 22:00 h) and L. cardium (peak abundances at 06:00 h) competed for three host fish species; a fourth species that they reportedly share (Pomoxis annularis (White Crappie)³³) was not in the survey (Table 2). One of these host fish species (P. flavescens) is also used by a third unionid species (E. dilatata – peak abundance at 20:00 h), which indicates the intensity of competition and the potential selective pressure on this system⁵⁴. Competition for hosts may drive unionids to parasitize lower quality hosts (i.e., those that provide low rates of juvenile mussel development and excystment³⁵ leading to reduced recruitment and population declines. In the extreme case of a highly successful invasive species in the Sydenham River, Neogobius melanostomus²⁷ is a very poor-quality host that is considered a sink for glochidia, which could lead to reproductive failure in unionids⁵⁶.

Other factors can exacerbate the competition for host fishes that mussels attempt to overcome. For example, as indicated above, the availability of space on the gills of the host may be limited (i.e., only ten glochidia were found on a host fish in the wild³⁴). Rejection by the host fish’s immune system due to immunity acquired from prior infections poses additional challenges^21,22 and acquired immunity is heightened by the age of the fish, multiple infestations and/or several high glochidia infection loads; outcomes that are likely heightened when hosts are limited^22,57. Unionids that release in the spring and early summer may induce the host’s acquired immunity before species that release glochidia in the late summer/fall period, further adding to this competition.

Regardless, there are several reasons why within-day temporal partitioning may be advantageous for this taxon beyond the reduction of competition for host fish^34,53. As revealed in this study, many species are coupled to specific host fish species on which they can reproduce successfully (Figure S6), i.e., their glochidia can successfully infest and successfully metamorphose into juveniles^35,58. Consequently, there is an advantage to matching glochidia abundance to the diel activity patterns of their host species, otherwise the likelihood of successful reproduction would be reduced⁵⁶. Secondly, as long-lived iteroparous species²¹, unionids may not need to reproduce successfully each season to maintain populations, and this may allow them to overcome issues such as space limitation on gills³⁴, variation in host fish abundance²², and acquired immunity³², which would be considered ephemeral over the multiple year/decades-long lifespan of a unionid²².

These results also have implications for the recovery, conservation and management of unionids, especially Species at Risk (SAR) unionids, which have experienced a variety of negative effects due to commercial exploitation, invasive species, and human-related pollution and land-use changes^21,26–28. Any activity that increases light and/or alters natural diel cyclic patterns of mussels or their hosts can interfere with mantle displays, disrupt mussel-host interactions and the timing of glochidia release³⁹. Consequently, results suggest that preserving timing signals for mussels and preventing disturbances that may alter the diel patterns of mussels and their hosts should be reduced at peak periods of glochidia abundance–i.e., during crepuscular and nocturnal periods in the late summer/fall^39,43 to protect these taxa. Future research should focus on other periods of glochidia release (i.e., spring and summer) to determine whether such precautions should also be applied. It should also include sampling fish for glochidia encystment simultaneously with the sampling of stream drift to compare glochidia release and successful encystment among competing species.

Conclusions

This study revealed that the glochidia of different species are not uniform in their abundance at any given time-of-day. Rather, glochidia abundance matched that of their host fish species and when several mussel species shared the same host, they exhibited a pattern that minimizes overlap with the other unionids. This allochrony of peak glochidia abundance among species is a specialization that mussels employ to attempt to partition their fish hosts temporally, which can likely be hindered by a variety of mechanisms employed by their fish hosts.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SLS: conceptualization, methodology, investigation, visualization, original draft, review and editing; TJM: methodology, review and editing; JDA; conceptualization, methodology, investigation, visualization, funding acquisition, project administration, supervision, review and editing.

Funding

Support for this research was provided by Fisheries and Oceans Canada (DFO; Species at Risk Program and SARNet [Freshwater Species at Risk Network]), and the Natural Sciences and Engineering Research Council of Canada (Discovery) to JDA.

Data availability

Data reported in this paper can be accessed in the Dryad Digital Repository: [http://datadryad.org/share/ffb8abe0C8Aw6aBABaEqOcbOKHDgVL5z6Uv6PkuLiJs](http:/datadryad.org/share/ffb8abe0C8Aw6aBABaEqOcbOKHDgVL5z6Uv6PkuLiJs) (Ackerman and Smodis, 2025).

Declarations

Competing interests

The authors declare no competing interests.