My research focuses on the evolution of North American water mites in the genus Arrenurus, Subgenus Micruracarus. In this presentation, I discuss why I chose to study these little known critters, and present some preliminary findings. Please contact me for more info.

1. EVOLUTION
OF
NORTH AMERICAN
MICRURACARUS
WATER MITES
R A C H E L S H O O P
G R A D U AT E S T U D E N T, D E PA R T M E N T O F B I O L O G Y
S A N D I E G O S TAT E U N I V E R S I T Y

2. MITES ARE EXTREMELY DIVERSE
• Worldwide, mite species rival beetles species in number.
• It is estimated that there are between 0.5 and 1 million total
species of mites .
– About 55,000 of these species have been described.
• Mite have various functions in their respective habitats.
– Some mites are parasitic for all or part of their lives, attaching to and
feeding on a host organism.
– Other mites are free-living. These mites may be predators,
herbivores, or detritivores (consuming decaying material).
Photos from top to bottom: Cranefly with larval mites (Martin Salt), red velvet mite (Sue
Carnahan), and Demodex folliculorum (human facial mite) (Letícia Satsiê Fátima de Freitas
Yamashita)

3. THE WATER MITES
• Also known to scientists as Hydrachnidia, these mites make up one of the largest
groups with more than 6,000 species (10% of all described mite species!)
• Can be found in nearly any aquatic habitat on all continents with the exceptions of
some marine shore waters and Antarctica (so far at least)
• Smith et. al. (2010) estimated that one square meter of littoral, vegetated substratum
in lakes may contain up to 2000 individuals representing 75 species in 25 or more
genera.

4. WHY STUDY MITES?
• Host/parasite relationships
– Mites can transmit disease
• Examples: Rikettsialpox, Scrub typhus
– Infestations of mites can harm the host
• Examples: Scabies, and scaly leg
– Potential bioindicators
• Because mites are so abundant and may have tight relationships to their
host species, they can be used to estimate the quality of the habitat.
• Basic biodiversity
– Due to their numbers and the diverse ecological roles mites serve, they
are an important part of the ecosystem. The more we know about
mites, the better we can understand the larger picture.
Legs of a hen with scaly leg.
The scales become loose due
to mites burrowing
underneath.
Image source: (Top) http://www.yourchickens.co.uk/care-and-advice/looking-good-part-ii-1-3581928; (Bottom) Paul Maier.
Damselfly with
larval mites.

5. • Life histories for water mites are also very diverse.
• Generally, the life cycle is as follows:
– Egg
– Larva (6 legs and hungry for food)
– Nymphochrysalis (Think of a butterfly’s chrysalis, but with a beautiful mite inside)
– Deutonymph (8 legs, still hungry for food)
– Imagochrysalis (Yep, mites are cool enough to have 2 chrysalis stages)
– Adult (8 legs and sexually mature… probably still hungry)
* Not all water mites go through all of the stages described above.
** Some species may be parasitic during only part of their life cycle. This is
demonstrated on the following slide.
WATER MITE LIFE HISTORY

6. WATER MITE LIFE HISTORY
Above photos by Jerry Evans
Illustration from Smith et. al., 2010; redrawn
and modified from Smith 1976

7. GENUS ARRENURUS
Divided into 4 North American Subgenera by Karl Thon in 1900.
• Described by male body shape, particularly concerning the cauda, a
male structure that projects from the posterior of the mite.
Males pictured to the right, from top to bottom:
– Arrenurus: Robust cauda with pygial lobes and a petiole
– Megaluracarus: Long cauda
– Truncaturus: Very short cauda
– Micruracarus: Short cauda with medial cleft
• Females are morphologically conserved. Thus, they are often identified by
association to male.
Image credits: Truncaturus male by Centre for Biodiversity Genomics Photography Group

8. MALES AND FEMALES
• Mites in the genus Arrenurus are sexually dimorphic.
– Males are typically smaller than females, with a structure
called a cauda projecting off of their posterior.
– Females are usually round or oval with a large genital
opening called a gonopore.
• Courtship methods differ among species, but male
Arrenurus typically use a glue secreted from glands on
their cauda to attach to the female (see right).
– This glue is very sticky, and can keep a mating pair
bonded for several hours.
Gonopore
Image credit: Arrenurus male and female by Gerard Visser

9. BUILDING OFF OF PREVIOUS HYPOTHESES
Phylogenetic relationships had only been speculated
by comparing morphology until very recently.
• In 2015, Więcek et. al. constructed a molecular
phylogeny using 2 genes
– CO1 (mitochondrial gene)
– 28S (nuclear ribosomal gene)
• This study focused primarily on European species of
Arrenurus, only 2 species from North America were
included in this phylogeny.
• Results appear to support the long-held hypothesis
that Micruracrus is not monophyletic (shown to the
right in blue).
Micruracarus

10. RESEARCH GOALS
1. Construct a robust molecular phylogeny of North American
Micruracarus using mitochondrial and nuclear markers
2. Combine the molecular phylogeny with a multimetric
morphological data set to answer two questions:
1. Can the characters that have historically been used to identify species
reliably predict species relationships?
2. What characters can be used to most accurately identify female
Micruracarus to species?
?

11. SPECIMEN COLLECTION
• Mites were collected from several sites across Ontario
(Canada), Florida, and Washington.
• Additional samples were donated from Alberta,
Canada, the Netherlands, and Poland.
• Mites were sampled from lakes, streams, and ponds
using a D-ring net with Nitex mesh.
– The mites and other small organisms were separated
from the collection bolus using fine sieves.
– Mites could then be collected from the rest of the
sample by hand in a pan or dish partially filled with
water.
• This allows the mites to swim out from any remaining
debris.

12. SPECIMEN PREPARATION
• Specimens were identified to species using a dissection microscope.
• All mites were stored in 95% ethanol in a -20˚C freezer.
• Photographs of dorsal, ventral, side views of specimens were captured using a BK Lab
Imaging System (Dun Inc.)
– For males, photos of the posterior (showing detail of the cauda) were also taken.
– All of these photos were later used to collect distance measurements and geometric
morphometric data for later analyses.

13. MOLECULAR METHODS
• DNA was extracted from specimens using a modified version of the
phenol/chloroform extraction protocol described by Palumbi et. al. 2002.
• After extraction, DNA was quantified using a Qubit dsDNA High Sensitivity Assay kit
(ThermoFisher Scientific).
• CO1 was amplified using PCR with the forward and reverse primer described by
Folmer (1994).
– Sequencing was carried out by Eton Biosciences.
• UCEs were obtained using the protocol described by Faircloth et. al. (2012).
– Arachnid probe set was designed by Starrett et. al. (2016).
– Sequencing was carried out using an Illumina HiSeq2500 with paired-end 125bp reads at
the DNA Sequencing Center at BYU.

14. CO1 (CYTOCHROME C OXIDASE 1)
• CO1 is a mitochondrial gene – nicknamed the “barcoding gene” – that has achieved
popularity for its efficiency in identifying specimens to species.
• Because it is a mitochondrial gene, however, the gene is only passed to offspring
from the mother. For this reason, cases of hybridization and introgression may go
unnoticed (Hebert et. al. 2003).
• A 659 bp portion of CO1 was included in this research because of its widespread use
for species identification.
– This eases comparison to other mite research.

15. ULTRACONSERVED ELEMENTS (UCES)
• UCEs are regions of DNA that, as the name suggests, are conserved, meaning the
sequences don’t vary very much across species.
– It is not yet known what the functions of many UCE regions are, but it is hypothesized that they
are involved in essential cell functions like gene regulation.
– Because these functions are so important, the sequences are limited to very little change.
• Fortunately, UCEs are flanked by regions on increasing diversity.
– Faircloth et. al. (2012) developed a method to probe for conserved regions in order to capture
the variable areas.
Starrett et. al., 2016
• The number of regions obtained from the
Faircloth et. al. method greatly increases the
number of informative characters analyzed by
phylogenetic inference software
– More data  Greater phylogenetic resolution

16. BIOINFORMATIC METHODS
CO1
• Sequences were assembled and
manually edited using Sequencher
v5.0 (Gene Codes Corporation,
MI)
• Alignments produced using
Clustal Omega (Sievers et. al.,
2011; Goujon et. al. 2010)
UCEs
• Raw read data processed using
Phyluce pipeline (Faircloth, 2015)
• Assemblies obtained using Trinity
• Aligned loci with using MAFFT and
edge-trimmed with GBLOCKS
• 75% and 90% complete data matrices
analyzed using RAxML
Both data sets were independently analyzed using RAxML (maximum likelihood)
• GTR+I+Γ model (JModel Test, v. 2.1.10)
• 1000 bootstraps replicates

17. Subgenus
Arrenurus
Subgenus Micruracarus
A. acutus/bicaudatus
complex
CO1 phylogeny
Maximum likelihood
tree (RAxML)
Subgenus
Megaluracarus
Subgenus Micruracarus (European sp.)
Subgenus Micruracarus
A. setiger group and
A. lyriger group
Subgenus Truncaturus
= 100 bootstrap support
= 85 - 99 bootstrap support
? = Arrenurus sp.
?
?
CO1 phylogeny suggests
polyphyly for several of the
Arrenurus subgenera.
Micruracarus split into 3 groups:
1. A. acutus/bicaudatus complex
2. European Micruracarus
3. North American Micruracarus
(A. setiger + A. lyriger groups)

18. CO1 phylogeny
ML tree (RAxML)
= 100 bootstrap support
= 85 - 99 bootstrap support
A. nsp. nr. acutus 1
A. nsp. nr. acutus 3
A. infundibularis
A. setiger
UCE phylogeny
ML tree (RAxML)
A. nsp. nr. acutus 2
UCE phylogeny shows increased
support for internal nodes compared to
CO1 phylogeny.
A. infundibularis clusters with North
American clade (A. setiger) in UCE
phylogeny.
Lines indicate
individuals represented
in both phylogenies.

19. MORPHOLOGY PRELIMINARY RESULTS
• Distance measurements are the most common method historically used to quantify
physical differences between water mite species. The distances measured from
specimens are illustrated below.
• Additional characters collected for males: Cauda width and cauda length
• All distances obtained using Adobe Photoshop CC 2018 ruler tool.
*Measurements
highlighted with blue
used to normalize data
set for body size.
Arrenurus (Micruracarus) crenellatus, female

20. • Among females, there is enough variation among the distance measurements analyzed
for some species to be distinguished.
• Species that are particularly difficult to identify to species (e.g. A. lyriger and A.
haitocaudatus) cannot be distinguished using the distance measurement data set.
Species
DISTANCE MEASUREMENT PRELIMINARY
RESULTS (FEMALES)

21. DISTANCE MEASUREMENT PRELIMINARY
RESULTS (MALES)
• Variation among males of different species allows most of them to be easily sorted
according to distance measurements (shown by PC1 and PC2).
• The variance illustrated by PC3 and PC4 appear to indicate individual differences with
species.
Species

22. GEOMETRIC MORPHOMETRICS
A geometric morphometric data set allows differences in shape to be quantified among
specimens using discrete points (landmarks) or curves (semilandmarks).
This is achieved by conducting a Generalized Procrustes Analysis (GPA), which accounts for
variation among samples due to differences in size and position by overlaying landmarks
collected from each specimen around a central point or centroid.
GPA includes 3 steps:
1. Translation (identifying a common centroid)
2. Rotation (for positional differences)
3. Scaling (for size differences)
Image source: Dr. Donald A. Jackson (http://jackson.eeb.utoronto.ca/procrustes-analysis/)

23. LANDMARKS AND SEMILANDMARKS
Semilandmarks include the margin of the dorsal
plate and acetabular plate (two features commonly
used to differentiate between females).
Images were digitized using tpsUtil (Rohlf 2003) and
landmarks and semilandmarks were collected using
tpsDIG2 (Rohlf 2004).
Mite landmarks include the locations of glands on
the dorsum and venter, locations of features along
the midline of the body, and points where ventral
plates converge.
Locations of glands on the cauda are also collected
for males.
A. (Micruracarus) laticaudatus, male
A. (Micruracarus) crenellatus, female

24. CONCLUSIONS & FUTURE DIRECTIONS
• Preliminary analysis suggests North American
Micruracarus are not monophyletic
– UCEs will be used to resolve questionable
relationships.
– Second batch of UCE samples have been sent
off for sequencing (Currently waiting for results).
• PCA indicates distance measurements of
morphological characters can be used to
distinguish some species.
• Landmark and semilandmark data collection
and analysis currently in progress.
• Further analysis is underway to test the
repeatability of positioning the mites for
photos.
CO1 UCE

25. ACKNOWLEDGEMENTS
Funding:
• Society for Integrative and Comparative Biology
– Libbie Hyman Memorial Scholarship
• San Diego State University
– Frank Alverson Memorial Scholarship
– Harold and June Grant Memorial Scholarship
Thesis committee members:
• Dr. Andrew Bohonak
• Dr. Marshal Hedin
• Dr. Stephen Schellenberg
• Dr. Bruce P. Smith – Ithaca College
Specimen contributions:
Monica Young – University of Guelph
Dr. Harry Smit
Dr. Andrej Zawal
Dr. Heather Proctor
Assistance with data collection and analysis:
Paul Maier
Shahan Derkarabetian
Erik Ekdale
Megan Smallcomb

26. PCA RESULTS
Female data set
Male data set
Character PC1 PC2 PC3 PC4
Dorsal Plate Length -0.1537875 0.45487278 0.06227894 -0.3415438
Dorsal Plate Width -0.2017054 0.43831777 0.03131758 -0.2664249
Cauda Width -0.2819887 -0.2601098 0.26452822 0.03337476
Cauda Length -0.081044 -0.279952 0.45486899 0.30774883
Coxal Plate II Diagonal -0.3629032 0.01675309 -0.2164756 0.12951247
C.P. III, IV Margin -0.3725005 0.0329738 -0.1849736 -0.0537511
C.P.III Diagonal -0.3784692 0.0596965 -0.1121574 0.01544908
C.P.IV Diagonal -0.3607061 -0.1300885 -0.1137511 0.14011368
C.P.I Margin to Gonopore -0.0687351 0.40402594 0.23006259 0.52535801
C.P. III, IV Gap Width -0.0470497 0.04237747 0.6019116 -0.3459158
C.P. I to Acetabular Plate -0.197071 0.39898256 0.19345891 0.30626521
Gonopore Length -0.3115243 -0.1614753 -0.1684209 -0.284317
A.P. Length -0.2191617 -0.1951201 0.36110568 -0.2783944
Side Max Depth -0.3389999 -0.2114649 -0.0157621 0.17091655
Character PC1 PC2 PC3 PC4
Dorsal Plate Length 0.26504899 -0.2312394 0.26674061 -0.2375231
Dorsal Plate Width 0.27928994 -0.0637424 0.19388207 -0.212554
Coxal Plate II Diagonal 0.29027352 -0.0343674 0.04450863 -0.1171058
C.P. III, IV Margin 0.28981778 -0.0689397 -0.0231694 -0.0786478
C.P.III Diagonal 0.28486516 -0.1391696 -0.0372364 -0.0821753
C.P.IV Diagonal 0.28992379 0.05270613 0.03582647 0.04528391
C.P.I Margin to Gonopore -0.1589021 -0.4787052 -0.3217666 -0.2463982
C.P. III, IV Gap Width 0.09821276 -0.4962631 0.33623735 0.47611468
C.P. I to Acetabular Plate -0.0792622 -0.5867144 -0.3514522 -0.098276
Gonopore Length 0.24914999 0.01640589 -0.4579524 -0.0968768
Gonopore Width 0.21991156 0.24863909 -0.4854181 0.21511748
A.P. Length 0.29366196 0.07747349 -0.1657417 0.07919773
A.P. Width 0.27636575 0.07374423 -0.0474991 -0.0854327
A.P. Upper Diagonal 0.24855771 -0.0841828 0.04571056 0.52384296
A.P. Lower Diagonal 0.25212415 0.03967727 0.23725574 -0.439107
Side Max Depth 0.28055537 -0.1119099 -0.1118305 0.19022992
PC1 PC2 PC3 PC4 PC5 PC6 PC7
Standard deviation 3.2083 1.44720.963240.779630.715240.59042 0.5168
Proportion of
Variance 0.6433 0.13090.057990.037990.031970.02179 0.0167
Cumulative
Proportion 0.6433 0.7742 0.83220.870190.902160.92395 0.9406PC8 PC9 PC10 PC11 PC12 PC13
Standard deviation 0.43463 0.38710.365660.35506 0.33720.31023
Proportion of
Variance 0.011810.009370.008360.00788 0.00710.00602
Cumulative
Proportion 0.952450.961820.970180.97806 0.98520.99118
PC1 PC2 PC3 PC4 PC5 PC6 PC7
Standard deviation 2.4601 1.737 1.34490.977490.780270.636670.58221
Proportion of
Variance 0.4323 0.2155 0.12920.068250.043490.028950.02421
Cumulative
Proportion 0.4323 0.6478 0.7770.845230.888710.917670.94188PC8 PC9 PC10 PC11 PC12 PC13
Standard deviation 0.50170.412530.336340.31843 0.29250.25849
Proportion of
Variance 0.017980.012160.008080.007240.006110.00477
Cumulative
Proportion 0.959860.972010.980090.987340.993450.99822

27. LITERATURE CITED
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an ultraconserved element probe set designed for Arachnida. Molecular Ecology. doi: 10.1111/1755-0998.12621.
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