Morphology of Genitalia and Non-Genitalic Contact Structures in Trouessartia Spp

Canadian Journal of Zoology

Morphology of genitalia and non-genitalic contact structures in Trouessartia spp. feather mites (Astigmata: Analgoidea: Trouessartiidae): is there evidence of correlated evolution between the sexes?

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2019-0291.R1

Manuscript Type: Article

Date Submitted by the 23-Jun-2020 Author:

Complete List of Authors: Byers, Kaylee; The University of British Columbia, Interdisciplinary Studies; Canadian Wildlife Health Cooperative, Animal Health Centre Proctor, H.C.;Draft University of Alberta, Department of Biological Sciences

Is your manuscript invited for consideration in a Special Zoological Endeavors Inspired by A. Richard Palmer Issue?:

Acariformes, COEVOLUTION < Discipline, feather mite, GENITALIA < Keyword: Organ System, Trouessartia, sexual conflict, sexual dimorphism

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Morphology of genitalia and non-genitalic contact structures in

Trouessartia spp. feather mites (Astigmata: Analgoidea: Trouessartiidae): is

there evidence of correlated evolution between the sexes?1

Kaylee A. Byers1a and Heather C. Proctor1

1 Department of Biological Sciences, University of Alberta, Edmonton, AB,

Canada

Emails:

Kaylee Byers: [email protected]

Heather Proctor: [email protected]

Correspondence:

Kaylee Byers, email: [email protected], phone: 778-980-9948

Department of Interdisciplinary Studies

University of British Columbia

270, 2357 Main Mall, H.R. MacMillan Building

Vancouver, BC, V6T 1Z4

a Current affiliations for Kaylee Byers are: Department of Interdisciplinary Studies, University of British Columbia, Vancouver, BC, Canada Biodiversity Research Centre, University of British Columbia, Vancouver, BC, Canada

1This article is one of a series of invited papers arising from the symposium “Zoological Endeavours Inspired by A. Richard Palmer” that was co-sponsored by the Canadian Society of Zoologists and the Canadian Journal of Zoology and held during the Annual Meeting of the Canadian Society of Zoologists at the University of Windsor, Windsor, Ontario, 14–16 May 2019. 1

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Morphology of genitalia and non-genitalic contact structures in

Trouessartia spp. feather mites (Astigmata: Analgoidea: Trouessartiidae): is

there evidence of correlated evolution between the sexes?

Kaylee A. Byers and Heather C. Proctor

Abstract

Positive correlations between the shapes of male and female sexual structures can be interpreted as cooperative or as combative. In the feather mite genus

Trouessartia Canestrini, 1899, the spermaducts of females range from entirely internal to extending externally for varying lengths, while male primary genitalia range from gracile to massive. Males also possess a pair of adanal suckers used to hold onto the dorsalDraft surface of the female during copulation. In the area of this attachment, females exhibit ornamentation and have strongly developed dorsal setae (setae h1), which we hypothesized serve to weaken the male’s hold during copulation. In male and female Trouessartia from 51 bird species, we compared female external spermaduct length and male genitalic

‘massiveness’ and explored whether patterns of female dorsal ornamentation and/or h1 seta size correlate with male adanal sucker size. Our results indicate that females with longer external spermaducts are associated with males with relatively massive genitalia. However, we found no significant relationship between male adanal sucker size and female ornamentation or h1 seta size.

Further information regarding how the genitalia interact during sperm transfer is necessary to interpret correlations in genitalia size and strong intersexual differences in dorsal ornamentation and seta size in Trouessartia.

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Keywords: Acariformes, coevolution, feather mite, genitalia, Trouessartia,

sexual conflict, sexual dimorphism

Introduction

In sexual species, both males and females have a vested interest in the fitness

gained from the successful completion of mating; however, reproductive

investment is often disproportionate between the sexes (Bateman 1948). As a

result, the sex with higher gametic or parental investment will often be more

selective of its mating partner (Parker et al. 1979). Females commonly invest

more in their offspring than do males (e.g., anisogamy, Bateman 1948); given

this, females are often the limitingDraft sex (Trivers 1972). This differential

investment between the sexes can promote sexual conflict, whereby each sex

acts to further its own interests. In some cases this struggle to gain control of

fertilization can be at the cost of the opposite sex (Parker et al. 1979; Arnqvist

and Rowe 2005; Rönn et al. 2007; Madjidian et al. 2012). Costs to females from

undesired matings include reduction in their own reproductive success

(Alexander et al. 1997), damage to the reproductive tract (Siva-Jothy 2006) and

increased predation (Rowe 1994).

Male genitalia and structures associated with holding or restraining

females are among the most rapidly evolving features in internally fertilizing

animals and are often more interspecifically variable than female genitalia

(Eberhard 1985; but see Simmons and Fitzpatrick 2019). This rapid

diversification in male genitalia is hypothesized to arise via selection to reduce

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hybridization (lock-and-key, Masly 2012) or via sexual selection (Eberhard

2010a) acting through cryptic female choice (Eberhard 1985), male-male competition for fertilization (sperm competition, Parker 1970), or sexually antagonistic coevolution (Arnqvist and Rowe 2005). The importance of selection against hybridization has fewer proponents today than when it was first suggested in the 1800s (Masly 2012), and the majority of current studies of genitalic evolution focus on disentangling the various sexual selection hypotheses. Although these hypotheses are not always mutually exclusive

(Hosken and Stockley 2004; Eberhard 2010b), there is growing evidence of the importance of conflict between the sexes in the evolution of reproductive features. Sexually antagonistic coevolutionDraft in reproductive structures has been documented in both vertebrates (Brennan et al. 2007) and invertebrates

(Arnqvist and Rowe 2005; Koene and Schulenburg 2005; Perry and Rowe 2012;

Bilton et al. 2016) and has been associated with traumatic insemination and harmful male genitalia in arthropods (Rönn et al. 2007; Tatarnic and Cassis

2010; Kamimura 2012; Dougherty et al. 2017).

In addition to sexually antagonistic coevolution in genitalic structures, sexual conflict may also influence non-genitalic contact structures involved in mate acquisition (Arnqvist and Rowe 2002a; Crumière et al. 2019). These structures can range from the sucker-like bursa of male nematodes (Ahmad and

Jairajpuri 1981) to the cerci of male dragonflies (McPeek et al. 2009). Non- genitalic contact devices employed by males to grasp females often correspond to the dimensions of the female’s ‘receptive’ structures (Arnqvist and Rowe

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2002a; Huber 2003; McPeek et al. 2009; but see Byers and Proctor 2014). An

excellent example of antagonistic coevolution in grasping structures occurs in

some diving beetles (Coleoptera: Dytiscidae), where males possess tarsal

suction cups to grasp females and females have evolved modified dorsal

indentations (macropunctures) and setose furrows at these areas that weaken the

male’s ability to retain a strong grip (Bergsten and Miller 2007; Karlsson Green

et al. 2013; Bilton et al. 2016). In response, males have evolved even more

elaborate suction cup morphologies to counteract these female modifications.

Similar patterns have been reported in the male grasping and female anti-

grasping structures (dorsally pointing spines) of water striders (Hemiptera:

Gerridae) (Arnqvist and Rowe 2002Draftb).

Correlations between genitalic structures are often difficult to test due to

the primarily internal nature of most female genitalia. However, some parts of

the female genitalia of spiders and feather mites (Acari: Astigmata) are

sclerotized (Proctor 2003; Kuntner et al. 2016) and are readily visible through

the body wall in cleared or slide-mounted specimens, making them ideal for

studying genitalic traits. Although the genitalia of female spiders have been the

focus of a fair amount of research on sexual selection (e.g., Eberhard 2004;

Huber et al. 2005; Kuntner et al. 2009; Kuntner et al. 2016), feather mites have

been almost entirely overlooked in studies of genitalic evolution in both sexes

(but see Klimov et al. 2017). Similar to other astigmatan mites, most male

feather mites possess a sclerotized tubular or rod-shaped aedeagus (copulatory

organ), which females of most species receive in their copulatory pore. This

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pore connects to the female’s internal sclerotized spermaduct that in turn leads to the spermatheca (Popp 1967; Proctor 2003). In some species of Astigmata, the spermaduct has both an internal section and an external section that extends outside of the female’s body (see Klimov and Sidorchuk 2011). Members of the vane-dwelling feather mite genus Trouessartia Canestrini, 1899 (Analgoidea:

Trouessartiidae) are of particular interest as the spermaduct is entirely internal in some species, while for other species it extends from the female’s body terminus to various lengths (Santana 1976). In Trouessartia this external spermaduct is often supported by an interlobar membrane that extends between a pair of triangular lobes at the posterior of the hysterosoma (Santana 1976) (Fig. 1a).

Intrageneric variation in female Draftgenitalia is usually described as less pronounced than in males (Eberhard 1985; Huber 2010). However there has been increasing recognition that female genitalia can vary significantly (Polihronakis 2006;

Puniamoorthy et al. 2010; Simmons and Fitzpatrick 2019) and that studies evaluating female genitalic diversity may provide valuable insights into coevolution between the sexes (Sloan and Simmons 2019).

Mating in feather mites occurs with the male and female oriented in opposite directions (Popp 1967; Walter and Proctor 1999) with the male’s venter apposed to the female’s dorsum (Proctor 2003). In the majority of feather mites, insemination is achieved through insertion of the male’s aedeagus into the female’s copulatory pore and into the distal portion of her internal spermaduct

(Proctor 2003; Klimov et al. 2017); however, in some species with external spermaducts (e.g. Pterolichoidea: Crypturoptidae) it appears that the male

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instead receives the tip of the female’s spermaduct inside his own genital

opening (Gaud and Atyeo 1996). Whether Trouessartia species mate in a

fashion similar to the Crypturoptidae is unknown, although Santana (1976)

concluded that based on the lack of an obvious intromittent structure among the

various genitalic sclerites, Trouessartia males likely receive the female’s

external spermaduct inside their fairly complex genitalic apparatus (see Fig. 2).

Females of Trouessartia spp. not only often possess an external

spermaduct, but most also appear to have more elaborate dorsal ornamentation

than do their conspecific males. This ornamentation is principally composed of

lacunae (pits) of various sizes and shapes on the dorsal side of the posterior

hysterosoma (Fig. 1a), which is Draftin the approximate region of where the male’s

adanal suckers affix. It is also in this region that the female’s h1 setae are

located. Similar to the external spermaduct, the length and area of the h1 setae

vary dramatically from hair-like microsetae to spearhead-like macrosetae

(Santana 1976). The larger setae may also play a role in thwarting unwanted

mating attempts, similar to the upwards-pointing abdominal spines of some

female water striders (Arnqvist and Rowe 1995).

In this study we aim to answer three questions relating to male and

female morphology in Trouessartia. First, we hypothesize that the elongation of

the external spermaduct may have coevolved with the male’s genitalic apparatus

(either through female choice or antagonistically), and we therefore predict a

correlation between dimensions of male and female genitalia. Second, if female

ornamentation hinders male attachment, we hypothesize that the extent of dorsal

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ornamentation in females will positively correlate with male adanal sucker size.

Third, we also predict a similar relationship between the size of the adanal suckers and the size of the female’s h1 setae.

Materials and methods

Host sampling

Avian hosts were collected or sampled in the field or from natural history museums from Canada, Europe, Australia, China, the Philippines and the United

States (see Acknowledgements for names of the field researchers). Feather mites were removed from their host birds in various ways depending on the region of capture. Birds collected from ChinaDraft and the Philippines were mist-netted, euthanized, and mites and other symbionts were removed by eye from dead birds and preserved in 70% ethanol. For European-caught birds, feathers were plucked from live hosts and stored in 70% ethanol for later inspection and removal of mites. In Australia, bird specimens were sampled from either the

Western Australian Museum (WAM) in Perth or the Queensland Museum (QM) in Brisbane by one of the authors (HP). Birds from the WAM were sampled in two ways: for dry skins, bird bodies were ruffled over a sheet of white paper and the mites were removed with fine forceps and placed into 80% ethanol; if birds were preserved in ethanol, symbionts were removed from the bottom of the container using a pipette. Birds from the QM were sampled in a similar manner to the dry study skins at the WAM. Mites from most Canadian birds were from

Alberta. For these hosts (which were mainly window- and roadkills), bodies

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were stored in a freezer at -20°C until processing by HP or KB. Frozen birds

were thawed and symbionts were collected via washing. Each bird was placed

into a container with ~15 mL of 95% ethanol, ~10 mL of Palmolive dish

detergent and an adequate volume of water to submerge the bird. The birds were

massaged in the solution to ensure that symbionts were removed from the wing,

tail and body feathers. Each bird was rinsed over a Fisher-Scientific 53-μm

mesh sieve and the washing liquid was poured through the same sieve.

Symbionts were washed from the mesh sieve with 80% ethanol and then stored

in 75 mL screw-cap containers for a minimum of one week before sorting to

allow the symbionts to rehydrate and sink. We examined washings for mites

using a Leica MZ16 dissecting microscopeDraft at 10-25x magnification.

Mites belonging to the genus Trouessartia were removed from ethanol,

cleared for 12-48 h in 85% lactic acid and mounted in polyvinyl alcohol medium

(6371A, BioQuip Products, Rancho Dominguez, California). Slides were placed

on a 40ºC slide warmer for a minimum of 4 days. Once cured, each slide was

examined using a Leica DMLB compound microscope with differential

interference contrast at 200-400x magnification. There are approximately 125

described species of Trouessartia, primarily from passerine hosts but with a few

known from Piciformes (Mironov and Galloway 2019). Species-level

identifications were made using relevant literature (Santana 1976; Mironov

1983; OConnor et al. 2005; Carleton and Proctor 2010; Mironov and Galloway

2019); however, most individuals other than those from Europe proved to be

undescribed. Males and females from the same host were assumed to be

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conspecific, given that for most hosts (except for members of the swallow family, Hirundinidae) only a single species of Trouessartia occurs per host species (Santana 1976). A subset of specimens in particularly good condition were prepared for scanning electron microscopy by dehydration followed by gold-coating with a Nanotek SEMprep 2 sputter coater, and imaged using a

JEOL 6301F Field Emission Scanning Electron Microscope. We included some male and female pairs in copula among those prepared for SEM with the hope of observing how their genitalia interact (this is difficult to do with slide-mounted specimens because the pairs usually separate in the mounting medium). A list of the sampled hosts with taxonomic authorities and mite associates is provided in

Table 1. Draft

Measurements

To assess correlation of morphological characteristics between the sexes, we took digital images of male and female mites using Image Capture software

(Apple Computer, Cupertino, CA, USA) and a Canon PowerShot S40 attached to the Leica DMLB compound microscope. Images were taken at 200 and 400x magnification and were analyzed using ImageJ (https://imagej.nih.gov/ij/).

Morphological structures were outlined by hand in the Image J program, and subsequently measured using the Image J software. For structures that were paired (i.e., h1 setae, adanal suckers) or were numerous (i.e., dorsal ornamentation comprised of multiple lacunae), we obtained an average measure for each mite as detailed below. For both sexes we measured the following (Fig.

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1a): area of the hysterosonotal shield to give an estimate of body size; area and

proportion of the hysterosonotal shield covered with ornamentation; average

area of five haphazardly-selected lacunae oriented on the longitudinal axis and

five on the lateral axis of the hysteronotal shield (total N = 10); average length

and area of the h1 setae located dorso-posteriorly on the hysterosoma. For

females, we measured the length of the total external spermaduct, the length of

the posterior interlobar membrane and the length of the spermaduct extending

externally past this membrane Fig. 1a). For males (Fig. 1b, c), we measured the

area of the genitalic complex (Fig. 1c) and calculated the ratio of the genitalia to

the hysteronotal shield area, and areas of both adanal suckers from which we

calculated average sucker area (seeDraft Supplementary Tables S1 and S2 for raw

measures).

Statistics

All analyses were performed using R Studio version 1.0.153, 2009 -

2017 (Boston, MA, USA). We first tested for normality in our data using the

Shapiro-Wilk test which has been shown to be appropriate for many types of

distributions and sample sizes (Razali and Wah 2011). For data not conforming

to normality, we transformed the data by obtaining the natural log (ln). We

tested for significant differences between adult male and female morphological

features using paired t-tests for normally distributed data or Wilcoxon tests for

non-normally distributed data. We performed correlation analyses for male and

female morphological characters on untransformed data using the non-

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parametric Spearman’s rank correlation coefficient as several of our characters remained non-normally distributed after transformation. For correlation analyses between female external spermaducts post interlobar membrane and male genitalic size, we removed females with external spermaducts which were entirely within the interlobar membrane. Although it would be ideal to have controlled our analyses for phylogeny rather than using each species as an independent data point, there is no published phylogeny for the genus

Trouessartia and very few are represented in GenBank as named species (as of

19 Dec 2019, there were entries for 20 named species of Trouessartia in

GenBank, most of which are from Spain and Russia). Draft

Results

Measurements

Most individuals were between 400 and 500 µm in body length (Supplementary

Fig. S1). Female Trouessartia had significantly larger hysteronotal shields than conspecific males (n = 51; paired t-test: t50 = 10.47, P < 0.01; Supplementary

Fig. S2a). Females also had a greater proportion of their hysteronotal shield covered with ornamentation (Z = -6.15, P < 0.01; Fig. 5a), larger h1 setae area

(Z = -3.09, P < 0.01; Fig. 5b), and longer h1 setae (t50 = 5.49, P < 0.01;

Supplementary Fig. S2b), than males. The total length of the female external spermaduct (n = 51) ranged from 17.2 µm to 99.7 µm while the length of the spermaduct extending beyond the end of the interlobar membrane was 2.2 to

54.3 µm (n = 28 spp.; see Fig. 3 for examples). In Trouessartia males (n = 51),

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the area of the genitalic apparatus ranged from 251.5 to 2967.9 µm2 and from

1% to 8.5% of the hysteronotal shield size. Average adanal sucker area ranged

from 50.8 to 338.6 µm2.

Interspecific variation in male genitalia

Images produced by the light microscope revealed considerable variation in

genitalic size and shape among male mites (Fig. 4a, b; see also Supplementary

Fig. S1). The genitalic apparatus in all species was located between the levels of

trochanters III and IV. Males of some species had slender genitalia whereas

others had an apparatus almost as wide as it was long. Curvature of the genitalia

was evident in most specimens, Draftbut the degree of this curvature was dependent

upon the orientation of the individual on the slide. Because slide-mounted

specimens were cleared, light-microscope images revealed both internal and

external aspects of the genitalia. Scanning electron microscopy, which images

only surficial aspects, showed that most genitalic structures visible in light

microscopy are not external, but rather lie behind a pair of flaps (Fig. 4c, d). In

some SEM specimens part of the genitalic apparatus was extended out between

the flaps (Fig. 4e, f, and see Fig. 2).

Correlation analyses

The majority of measured structures had non-normal distributions for

both males and females, even after performing log transformations; therefore we

analyzed untransformed data using non-parametric tests. Hysteronotal shield

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size was strongly correlated for conspecific males and females (rs = 0.73, n = 51,

P < 0.01) indicating that larger females tended to be associated with larger males (Supplementary Fig. S2a). While female external spermaduct length was not significantly correlated with female hysteronotal shield size (rs = 0.08, n =

51, P = 0.55) (Supplementary Fig. S2c), the size of male genitalia was significantly positively correlated with male hysteronotal shield size (rs = 0.45, n

= 51, P < 0.01) (Supplementary Fig. S2d). Females with especially long external genitalia (> 75 µm) were collected from these host species: Emberiza spodocephala Pallas, 1766; Pyrrhula leucogenis Ogilvie-Grant, 1895; Geospiza fuliginosa Gould, 1837; and Geospiza magnirostris Gould, 1837

(Supplementary Table S1). MalesDraft with especially massive genitalia (>1900 µm2) were collected from these host species: Grallaria ruficapilla Lafresnaye, 1842;

Grallina cyanoleuca (Latham, 1802); G. fuluginosa; and Sialia sialis (Linnaeus,

1758) (Supplementary Table S2). Male genitalia size was correlated with the total length of the female external spermaduct (rs = 0.37, n = 51, P < 0.01) (Fig.

5c) as well as the length of the part of the spermaduct that extended past the terminal edge of the interlobar membrane (rs = 0.59, n = 28, P < 0.01) (Fig. 5d; smaller sample size because not all females had external spermaducts that extended past the interlobar membrane). In contrast, male sucker area was not correlated with the area of hysteronotal ornamentation on the female (rs = 0.17, n = 51, P = 0.22; Fig. 5e); nor did male sucker area correlate with either h1 seta area (rs = -0.04, n = 51; P = 0.78; Fig. 5f), h1 seta length (rs = -0.08, n = 51, P =

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0.57; Supplementary Fig. S2e) or average size of lacunae (rs = -0.09, n = 51, P =

0.52; Supplementary Fig. S2f).

Discussion

Correlations between male and female genitalia

Genitalic morphology, particularly that of males, has long been used in species

identification due to their tendency to diverge rapidly, a phenomenon variously

attributed to female choice (Eberhard 1985), male choice (Yoshizawa et al.

2018), intrasexual competition (Parker 1970), sexually antagonistic coevolution

(Arnqvist and Rowe 2005), or selection against hybridization (Masly 2012).

Male genitalia are often highly variableDraft while females appear to display

relatively little variation in genitalic morphology among closely related taxa

(Eberhard 1985). In the Trouessartia species we examined, males showed the

predicted interspecific variation in shape and size of the genitalic apparatus, but

females also demonstrated considerable variation in the length of the external

spermaduct. If the external spermaduct is involved in correlated evolution of

genitalic traits, then variation in its shape should correlate with some aspect of

male genitalia. In support of this hypothesis, we found that the total length of the

female external spermaduct and the length extending past the end of the

interlobar membrane were significantly positively correlated with male genitalic

size (Fig. 5c, d). However, the strength of these correlations (rs = 0.37 and 0.59,

respectively) indicates that much variation in the genitalia of both sexes is

unexplained. This may be because our simple measurements fail to capture the

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full nature of interspecific variation. Several studies evaluating genitalic correlations between the sexes have used indices of complexity (Kuntner et al.

2009; Tatarnic and Cassis 2010; Kuntner et al. 2016) or specific aspects of morphology associated with damaging and/or protective aspects of male and female genitalia (Rönn et al. 2007; Kamimura 2012; Dougherty et al. 2017).

Assessment of different aspects of the male’s genital region (e.g., pregenital apodeme, structure of the various internal sclerites) or of the female’s internal spermaduct and spermatheca might provide additional evidence for coevolution of male and female structures.

Extensions of the spermaduct that act as external copulatory tubes have been reported in numerous astigmatanDraft mite taxa including in some non-feather mite taxa such as Chaetodactylidae (e.g. Chaetodactylus osmiae (Dufour, 1839);

Klimov and OConnor 2008), Glaesacaridae (Klimov and Sidorchuk 2011) and

Rosensteiniidae (OConnor and Reisen 1978), and in the feather mite families

Caudiferidae (Gaud and Atyeo 1996), Crypturoptidae (Gaud et al. 1973),

Eustathiidae (Peterson et al. 1980), as well as several genera within the

Pterolichidae (Atyeo 1992) and Thoracosathesidae (OConnor 2009). Klimov and Sidorchuk (2011) suggest that copulatory tubes evolved through antagonistic interactions between the sexes and may allow females to reject undesired males. Klimov and Sidorchuk (2011) also suggest that the extended copulatory tubes in some feather mites may be indicative of precopulatory female choice, though they do not discuss how this might be accomplished. In feather mites, males often engage in precopulatory guarding of female

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tritonymphs (Witalińsky et al. 1992; Byers and Proctor 2014) and presumably

mate with the newly moulted adult female upon eclosion, which would appear to

minimize the female’s ability to actively select among potential mates.

Ascribing the selective force behind the evolution of externalized female

genitalia is difficult without studies of mating behaviour and functional

morphology (Brennan and Prum 2015). But even with very good understanding

of how male and female anatomy interact, Yoshizawa et al. (2018) were not able

to conclude whether the penis-like female genitalia of cave-dwelling bark lice

(Insecta: Psocodea) evolved via sexually antagonistic selection, female

competition for male nuptial gifts, male choice for stimulation, or some

combination of these forces. Draft

Differences in dorsal sculpturing and seta size between the sexes

We confirmed that female Troussartia have a significantly greater proportion of

their dorsal shield covered with lacunae (pits) than do the males. Similarly, the

postero-dorsal setae h1 of females were more strongly developed. Contrary to

our expectations, though, there was no correlation between the size of the male’s

adanal suckers and the degree of female ornamentation and h1 seta dimensions.

While this lack of correlation is consistent with the possibility that these

structures are not used to hinder male mating, their presence, regardless of

correlations in size, may still weaken the male’s grip on her dorsum prior to or

during sperm transfer. Although there are numerous studies that report a lack of

correlation in contact devices (e.g. Collembola in Eberhard 1985; Eberhard

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2004) variations in size and density of the dorsal lacunae of female Trouessartia spp. are reminiscent of the dorsal macropunctures present on females of some dytiscid water beetles (Bergsten and Miller 2007). Contrary to the correlated relationships between sucker adaptations and female dorsal morphology in dytiscid beetles, in Trouessartia spp., the variations in male sucker size do not match the extent to which females vary in dorsal ornamentation. While it is possible that these dorsal lacunae are “cooperative” instead of “antagonistic”

(Eberhard 2004), this is unlikely because the lacunae do not appear to allow for insertion or attachment by the male (Eberhard 2004). Indeed, in Trouessartia spp., the adanal suckers have an area significantly greater than the area of the largest dorsal lacunae of the female.Draft Further, as the adanal suckers attach through negative pressure (Witalińsky et al. 1992), it seems logical that these dorsal lacunae would serve not to encourage but rather to disrupt attachment as is seen in diving beetles (Bergsten and Miller 2007). Moreover, if dorsal lacunae are cooperative, we would still expect to see a correlation between these grasping structures (McPeek et al. 2009). It is possible that the lacunae in female

Trouessartia are not sexually selected features but rather are related to gas exchange. In both sexes of the highly sclerotized water mite genus Arrenurus

Dugès, 1834 (Prostigmata: Arrenuridae), the thick integument is dotted with many circles of lightly sclerotized cuticle covering gas-filled tracheal loops

(Mitchell 1972). Although astigmatan mites don’t have tracheal systems for gas exchange, the thin cuticle of the dorsal lacunae of female Trouessartia may allow oxygen to permeate into their ovary and developing egg (Trouessartia

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mature only one very large egg at a time, see ‘Pyrrhula leucogenis Ogilvie-

Grant, 1895’ in Supplementary Fig. S1).

A lack of correlation between adanal sucker area and the dimensions of

the female’s h1 setae is further indication that these contact structures may not

affect successful coupling; though they may influence copulation in a way that

was not measurable in our study. Eberhard (2004) suggests that resistance

structures that can be employed facultatively (e.g. mobile structures such as

erectable spines) work best in antagonistic interactions. The internal anatomy at

the base of the h1 setae in Trouessartia spp. is currently unknown; however, it is

possible that they have associated musculature that allows females to erect the

setae and impede coupling. Additionally,Draft glands and their secretions may play a

role in copulation. For example, phoretic deutonymphs in many uropodid mites

employ glandular secretions to attach to their host (Bajerlein and Witalínski

2012), and Fain et al. (1984) suggest that secretions may aid in male-female

coupling in fur mites (Astigmata: Chirodiscidae). Popp (1967) states that males

of a proctophyllodid feather mite secrete an adhesive substance from near their

adanal suckers that, in addition to the ‘vacuum’ action of the suckers

themselves, help the male to hold onto females and also “...smooths the dorsal

integument of the female...” . If glandular secretions are employed by adult male

Trouessartia to assist in affixing to females, then it is possible that the

elaborations in dorsal ornamentation in females are evolving in response to these

secretions rather than to surface area of suckers (e.g., secretions may have to

first fill up the lacunae before they are able to act as adhesives).

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Future considerations

Although there have been suggestions as to the ways in which male and female genitalia in Trouessartia interact (Santana 1976; OConnor 2009), it is still unclear as to whether the male receives the female’s spermaduct in his genital opening. In the Crypturoptidae, the male’s aedeagus has moved between the first set of coxae, and is believed to receive the female’s external spermaduct (Gaud and Atyeo 1996). Although such an extreme displacement of the male’s genitalic opening is not evident in Trouessartia spp., the male’s genitalia may still receive the female’s spermaduct. In our study we succeed in obtaining SEM images of only a single pair of adultsDraft in copula. Through SEM examination of this couple it appears that the male’s genital organ clasps the female external spermaduct between its two halves (Fig. 2), though whether or not the external spermaduct fits within a groove in the male’s aedeagus as suggested by

OConnor (2009) requires further examination. To fully understand the relationship between the female spermaduct and male aedeagus in Trouessartia spp., it would be ideal to observe live mites in copula. Similar observations would also help to elucidate whether aspects of mite behavior play a role in antagonistic interactions. Indeed, Brennan and Prum (2015) highlight the importance of understanding the behaviour of species to further our understanding of genital coevolution. This, however, is difficult as feather mites require their hosts to complete their life cycle (Clayton and Walther 1997) and mites removed from feathers are unlikely to behave naturally. Increased

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sampling may result in the serendipitous collection of additional mating pairs.

Similarly, studies evaluating male genitalic structure in greater detail (e.g., via

histological sections or transmission electron microscopy) should further

elucidate how the male aedeagus might interact with the female spermaduct.

Although Trouessartia species descriptions frequently include illustrations of

the spermatheca (e.g., Santana 1976), detailed drawings of the complex male

genitalia are rare in taxonomic literature (for an exception see Mironov and

Galloway 2019).

Some aspects of our study require verification. In particular, many of the

species we included are undescribed. This is not surprising, as Mironov and

Galloway (2019) estimate that atDraft most only 10% of the species in this genus

have been described. Most bird species host only one species of Trouessartia,

however Gaud and Atyeo (1986) have found that members of the swallow

family (Hirundinidae) can carry two species. Our analysis includes one species

within the Hirundinidae (Tachycineta bicolor (Vieillot, 1808)), and we were

able to identify the mites obtained from this bird to species allaying concerns

that males and females in this case might be from different species; however, in

the absence of taxonomic literature with descriptions of males and females, it is

possible that in some cases we treated male and female mites from a single host

as conspecifics when they were not. It is also possible that contamination

between different host species may have occurred in the field or in museums.

For birds mist-netted in the field, cross-contamination of symbionts between

hosts may have occurred if the same bags were used for temporarily holding

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birds. For birds stored in drawers or in ethanol in museum collections, cross- contamination can occur if birds are moved or reorganized, or if preservative is re-used. Also in need of investigation is the source of strong differences in morphology of mites identified to the same species but from different congeneric hosts (Trouessartia geospiza OConnor, Foufopoulos and Lipton,

2005, from two Geospiza spp. Gould, 1837). The morphological differences we observed may indicate that the mites are not conspecific. For all these reasons, further collection of the undescribed Trouessartia in our study is needed, together with morphological and molecular approaches to confirm conspecificity of males and females. Ideally, future investigations of morphological evolution in this groupDraft of mites should include a molecular phylogeny based on freshly collected specimens to allow for more sophisticated tests of coevolutionary hypotheses. At the moment, relatively few sequences are available for Trouessartia species, and most from a single project (Doña et al.

2015) with specimens from only two geographical regions (Spain and

Kalingrad). Byers (2013) addressed a similar set of questions with the same morphological dataset and used host bird phylogeny as a proxy for mite phylogeny. The phylogenetic approach showed the same patterns as the simpler correlational approach we employ in the present work; however, it is risky to assume that patterns of feather mite speciation precisely follow those of their hosts (e.g., Doña et al. 2015), so the corroborating results of Byers (2013) should be viewed with caution.

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Conclusions

In Trouessartia species, the length of the female’s external spermaduct

correlates with the overall size of the male’s aedeagus. Whether the elongation

of the female external spermaduct plays a role in female choice or sexually

antagonistic coevolution is unclear. However, our analyses of female

ornamentation and h1 seta size did not reveal any evidence for correlation of

these traits with the surface area of male adanal suckers and so these parts seem

not to have coevolved. To better determine the role of these morphological

features in sexual interactions, further investigation into the mating behaviour of

these mites is vital, as well as investigation of finer anatomical details of both

male and female genitalia and ofDraft the dorsal ornamentation and setae in females.

Given the diversity of female structures and their potential interactions with

male anatomy, Trouessartia is an ideal group for future studies on coevolution.

Acknowledgments

Thanks to Dr. Sarah Bush (University of Utah) for sending the mites from China

and the Philippines, Dr. Terry Galloway (University of Manitoba) for those from

Manitoba, and both Drs. Sofia Fernandéz and Ismael Galván (Centre National de

la Recherche Scientifique, Paris) for mites from Spain. Dr. Owen Seeman

(Queensland Museum) and Dr. Mark Harvey (Western Australian Museum)

kindly hosted HP on mite-collecting visits. We extend our thanks to Dr. Jeffrey

Newton and Brandon Doty for their assistance as well as to Drs. Jocelyn Hall,

Bruce Heming, and Richard Palmer for their feedback on an earlier version of

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this manuscript. We also thank two anonymous reviewers and the Guest Editor for their constructive feedback which improved the clarity and focus of this manuscript. Bodies of birds from Alberta were held under Alberta Sustainable

Resource Development and Canadian Wildlife Service permits to HP. This research was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant to HP.

Data Availability

Much of the original data for this manuscript is included in the supplementary material and any additional data are available directly from the authors upon request. Draft

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1 Figure Legends

2 Figure 1. Trouessartia Canestrini, 1899 feather mite body measurements (μm).

3 Both males and females were measured for the area of the hysteronotal shield (i)

4 and area of the hysteronotal shield containing ornamentation (ii). The average

5 size of lacunae along the longitudinal axis (iii) and lateral axis (iv) was

6 measured, as well as an average measurement for these two sets of lacunae

7 combined. Males and females were measured for the length and area of the

8 dorsal h1 setae (v). For females, we measured the total length of the female

9 external spermaduct (vi), the length of the interlobar membrane (vii) and the

10 length of the spermaduct extending past the posterior interlobar membrane (viii).

11 For males, we measured the areaDraft of the aedeagus (ix) and average area of the

12 ventral adanal suckers (x). Illustration drawn after Trouessartia geospiza

13 OConnor, Foufopoulos, and Lipton 2005 (OConnor et al. 2005) in Adobe

14 Illustrator CS3 (Adobe Systems, San Jose, CA).

16 Figure 2. Scanning electron micrograph of a male and female Trouessartia

17 bochkovi Mironov and Galloway, 2019 in copula. The view shows the ventral

18 side of the female on the left and the male on the right. It appears that the male’s

19 genital organ clasps the female’s external spermaduct between its two halves,

20 although how these organs interconnect remains unclear. Mites were collected

21 from Tachycineta bicolor (Vieillot, 1808).

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23 Figure 3. Scanning electron micrographs taken of the dorsal sides of female

24 Trouessartia spp. Canestrini, 1899 obtained from the avian hosts (a) Progne

25 subis (Linnaeus, 1758) (b) Pyrrhula leucogenis Ogilvie-Grant, 1895 and (c)

26 Hirundo rustica Linnaeus, 1758. Arrows indicate the external spermaduct.

28 Figure 4. Light microscopy images (a, b) and scanning electron micrographs

29 (SEM) taken of the male genital apparatus (GA), anus (A) and adanal suckers

30 (AdS). Light microscope images are taken of males from the avian host species

31 (a) Sialia sialis (Linnaeus, 1758) and (b) Stachyridopsis ruficeps (Blyth, 1847)

32 while SEM images are taken of mites removed from (c) Cyornis herioti Ramsay,

33 1886 (d) Pyrrhula leucogenis OgilvieDraft‐Grant, 1895 (e) Dicrurus balicassius

34 (Linnaeus, 1766) and (f) Turdus merula (Linnaeus, 1758). In figures (c) and (d)

35 the male genital apparatus appears to be enclosed behind a hatch-like cover,

36 while in figures (e) and (f) the genital sclerites are visible through the genital

37 opening.

39 Figure 5. Correlations in size of morphological features between adult male and

40 female Trouessartia spp. Canestrini, 1899. Females exhibited significantly

41 greater dorsal ornamentation (a) and larger h1 setae (b) than males. Females

42 with longer external spermaducts were associated with males with relatively

43 massive genitalia (c, d), while there was no significant correlation between

44 female ornamentation (e) or h1 seta area (f) with male adanal sucker size.

45 Correlations were performed on 51 conspecific pairs, except for 4(d) which was

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46 based on a subset of 28 pairs in which the female did have part of the external

47 spermaduct extending beyond an interlobar membrane. Where comparing the

48 same feature for both sexes (a, b), an orange trendline representing an exact 1:1

49 relationship is given for context.

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Table 1. Trouessartia spp. feather mites retrieved from 51 avian host species and their location of capture.

Host Taxonomy Capture Location Mite Taxonomy Family Species Taxonomic Authority Species Acrocephalidae Acrocephalus arundinaceus (Linnaeus, 1758) Ismael Galvon Trouessartia trouessarti Oudemans, 1904 Acrocephalus melanopogon (Temminck, 1823) Ismael Galvon Trouessartia cf. bifurcata (Trouessart, 1884) Cardinalidae Passerina cyanea (Linnaeus, 1766) Mount Berry, Georgia Trouessartia sp. Cotingidae Ampelioides tschudii (Gray, 1846) Cali, Colombia Trouessartia sp. Dasyornithidae Dasyornis brachypterus (Latham, 1802) Australia * Trouessartia sp. Dicruridae Dicrurus balicassius (Linnaeus, 1766) Aurora Memorial National Park, Trouessartia sp. Philippines Emberizidae Emberiza spodocephala Pallas, 1776 Shuipu village and Kuan Kuoshui Trouessartia sp. Nature Reserve, China Fringillidae Pyrrhula leucogenis Ogilvie-Grant, 1895 Aurora Memorial National Park, Trouessartia sp. Draft Philippines Grallariidae Grallaria ruficapilla Lafresnaye, 1842 Cali, Colombia Trouessartia sp. Hirundinidae Tachycineta bicolor (Vieillot, 1808) Alberta, Canada Trouessartia bochkovi Mironov & Galloway, 2019 Leiothrichidae Minla cyanouroptera (Hodgson, 1838) Kuan Kuoshui Nature Reserve, China Trouessartia sp. Meliphagidae Lichenostomus frenatus (Ramsay, 1874) Lake Eacham, Australia Trouessartia sp. Monarchidae Grallina cyanoleuca (Latham, 1802) Derby-West Kimberley and Victoria, Trouessartia sp. Australia † Motacillidae Motacilla cinerea Tunstall, 1771 Kuan Kuoshui Nature Reserve, China Trouessartia cf. jedliczkai (Zimmerman, 1894) Muscicapidae Brachypteryx montana Horsfield, 1821 Aurora Memorial National Park, Trouessartia sp. Philippines Cinclidium leucurum (Hodgson, 1845) Shiwandashan Nature Reserve, China Trouessartia sp. Copsychus luzoniensis (Kittlitz, 1832) Aurora Luzon Island, Philippines Trouessartia sp. Cyornis banyumas (Horsfield, 1821) Jing Xin County Nature Reserve and Trouessartia sp. Kuan Kuoshui Nature Reserve, China Cyornis hainanus (Ogilvie-Grant, 1900) Shiwandashan Nature Reserve, China Trouessartia sp. Cyornis herioti Ramsay, 1886 Angat, Philippines Trouessartia sp. Cyornis rufigaster (Raffles, 1822) Burdeos, Philippines Trouessartia sp. Enicurus leschenaulti (Vieillot, 1818) Kuan Kuoshui Nature Reserve, Canada Trouessartia sp. Erithacus rubecula (Linnaeus, 1758) Cádiz, Spain Trouessartia sp. Niltava davidi La Touche, 1907 Kuan Kuoshui Nature Reserve, China Trouessartia sp.

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Parulidae Geothlypis philadelphia (Wilson, 1810) Alberta, Canada Trouessartia sp. Oreothlypis peregrina (Wilson, 1811) Edmonton, Alberta, Canada Trouessartia sp. Seiurus aurocapillus (Linnaeus, 1766) Alberta, Canada Trouessartia sp. Setophaga petechia (Linnaeus, 1766) Barrhead, Edmonton, Hinton and Trouessartia sp. Millet, Alberta, Canada Setophaga ruticilla (Linnaeus, 1758) Alberta, Canada Trouessartia sp. Pellorneidae Alcippe morrisonia Swinhoe, 1863 Kuan Kuoshui Nature Reserve, China Trouessartia sp. Picidae Veniliornis cassini (Malherbe, 1862) Cali, Colombia Trouessartia sp. Veniliornis nigriceps (Orbigny, 1840) Cali, Colombia Trouessartia sp. Ptilonorhynchidae Sericulus chrysocephalus (Lewin, 1808) Australia * Trouessartia sp. Pycnonotidae Hypsipetes mcclellandii (Horsfield, 1840) Jing Xin County Nature Reserve, Trouessartia sp. China Regulidae Regulus ignicapillus (Temminck, 1820) Cádiz, Spain Trouessartia reguli Mironov 1983 Rhipiduridae Rhipidura albicollis (Vieillot, 1818) Jing Xin County Nature Reserve, Trouessartia sp. China Rhipidura cyaniceps (Cassin, 1855) Zabali Camp, Philippines Trouessartia sp. Sapayoidae Sapayoa aenigma Hartert,Draft 1903 Gamboa, Panama†; Cali and Rio Uva ‡ Trouessartia sp. Colombia Sittidae Sitta frontalis Swainson, 1820 Aurora Memorial National Park, Trouessartia sp. Philippines Sturnidae Sturnus vulgaris Linnaeus, 1758 Alberta, Canada Trouessartia rosterii (Berlese 1886) Sylviidae Lioparus chrysotis (Blyth, 1845) Kuan Kuoshui Nature Reserve, China Trouessartia sp. Sylvia atricapilla (Linnaeus, 1758) Cádiz, Spain Trouessartia bifurcata (Trouessart, 1884) Sylvia melanocephala (Gmelin, 1789) Cádiz, Spain Trouessartia inexpectata Gaud 1957

Thraupidae Geospiza fuliginosa Gould, 1837 Galapagos Trouessartia geospiza OConnor, Foufopoulos and Lipton, 2005 Geospiza magnirostris Gould, 1837 Galapagos Trouessartia geospiza Timaliidae Pomatorhinus montanus Horsfield, 1821 Bali, Indonesia † Trouessartia sp. Stachyridopsis ruficeps (Blyth, 1847) Kuan Kuoshui Nature Reserve and Trouessartia sp. Shuipu Village, China Turdidae Catharus ustulatus (Nuttall, 1840) Edmonton and Ministik Hills, Alberta, Trouessartia sp. Canada

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Sialia sialis (Linnaeus, 1758) Georgia, USA Trouessartia sialiae Carleton and Proctor 2010 Turdus merula Linnaeus, 1758 Cádiz, Spain Trouessartia incisa Gaud 1957 Tyrannidae Tyrannus tyrannus (Linnaeus, 1758) Alberta and Manitoba, Canada Trouessartia sp.

* Indicates specimens collected from the Queensland Museum, Australia † Indicates specimens collected from the Western Australian Museum, Australia ‡ Indicates specimens collected from the American Museum of Natural History, New York Note: Currently undescribed species are listed by genus.

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Figure 1. Trouessartia Canestrini, 1899 feather mite body measurements (μm). Both males and females were measured for the area of the hysteronotal shield (i) and area of the hysteronotal shield containing ornamentation (ii). The average size of lacunae along the longitudinal axis (iii) and lateral axis (iv) was measured, as well as an average measurementDraft for these two sets of lacunae combined. Males and females were measured for the length and area of the dorsal h1 setae (v). For females, we measured the total length of the female external spermaduct (vi), the length of the interlobar membrane (vii) and the length of the spermaduct extending past the posterior interlobar membrane (viii). For males, we measured the area of the aedeagus (ix) and average area of the ventral adanal suckers (x). Illustration drawn after Trouessartia geospiza OConnor, Foufopoulos, and Lipton 2005 (OConnor et al. 2005) in Adobe Illustrator CS3 (Adobe Systems, San Jose, CA).

182x96mm (300 x 300 DPI)

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Figure 2. Scanning electron micrograph of a male and female Trouessartia bochkovi Mironov and Galloway, 2019 in copula. The view shows the ventral side of the female on the left and the male on the right. It appears that the male’s genital organ clasps the female’s external spermaduct between its two halves, although how these organs interconnect remains unclear. Mites were collected from Tachycineta bicolor (Vieillot, 1808).

83x26mm (300 x 300 DPI) Draft

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Figure 3. Scanning electron micrographs takenDraft of the dorsal sides of female Trouessartia spp. Canestrini, 1899 obtained from the avian hosts (a) Progne subis (Linnaeus, 1758) (b) Pyrrhula leucogenis Ogilvie- Grant, 1895 and (c) Hirundo rustica Linnaeus, 1758. Arrows indicate the external spermaduct.

182x111mm (300 x 300 DPI)

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Figure 4. Light microscopy images (a, b) and scanning electron micrographs (SEM) taken of the male genital apparatus (GA), anus (A) and adanal suckers (AdS). Light microscope images are taken of males from the avian host species (a) Sialia sialis (Linnaeus, 1758) and (b) Stachyridopsis ruficeps (Blyth, 1847) while SEM images are taken of mites removed from (c) Cyornis herioti Ramsay, 1886 (d) Pyrrhula leucogenis Ogilvie‐Grant, 1895 (e) Dicrurus balicassius (Linnaeus, 1766) and (f) Turdus merula (Linnaeus, 1758). In figures (c) and (d) the male genital apparatus appears to be enclosed behind a hatch-like cover, while in figures (e) and (f) the genital sclerites are visible through the genital opening.

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Figure 5. Correlations in size of morphological features between adult male and female Trouessartia spp. Canestrini, 1899. Females exhibited significantly greater dorsal ornamentation (a) and larger h1 setae (b) than males. Females with longer external spermaducts were associated with males with relatively massive genitalia (c, d), while there was no significant correlation between female ornamentation (e) or h1 seta area (f) with male adanal sucker size. Correlations were performed on 51 conspecific pairs, except for 4(d) which was based on a subset of 28 pairs in which the female did have part of the external spermaduct extending beyond an interlobar membrane. Where comparing the same feature for both sexes (a, b), an orange trendline representing an exact 1:1 relationship is given for context.

171x236mm (300 x 300 DPI)

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