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Special Issue: Fossil and Modern Clam Shrimp (Branchiopoda: Spinicaudata, Laevicaudata)
Testing Weissman’s Lineage Selection Model
for the Maintenance of Sex: The Evolutionary
Dynamics of Clam Shrimp Reproduction over
Geologic Time
Timothy I. Astrop1,*, Lisa Park Boush2, and Stephen C. Weeks3
1Fossil Forest Project, Blast Road, Brymbo, Wales, United Kingdom, LI11 5BT. *Correspondence: E-mail: timastrop@gmail.com (Astrop)
2Department of Geosciences, University of Connecticut, Storrs, CT 06269-1045, USA. E-mail: lisa.park_boush@uconn.edu (Park Boush)
3Department of Biology, The University of Akron, Akron, OH 44325-3908, USA. E-mail: scw@uakron.edu (Weeks)
Received 11 June 2020 / Accepted 15 June 2020 / Published 5 August 2020
Special issue (articles 32-46) communicated by Thomas A. Hegna and D. Christopher Rogers
One of the most perplexing questions within evolutionary biology is: “why are there so many methods of
reproduction?” Contemporary theories assume that sexual reproduction should allow long term survival
as dispersal and recombination of genetic material provides a population of organisms with the ability
to adapt to environmental change. One of the most frustrating aspects of studying the evolution of
reproductive systems is that we have not yet been able to utilize information locked within the fossil record
to assess breeding system evolution in deep time. While the fossil record provides us with information on
an organism’s living environment, as well as some aspects of its ecology, the preservation of biological
interactions (reproduction, feeding, symbiosis, communication) is exceedingly rare. Using both information
from extant taxa uncovered by a plethora of biological and ecological studies and the rich representation
of the Spinicaudata (Branchiopoda: Crustacea) throughout the fossil record (from the Devonian to today),
we address two hypotheses of reproductive evolutionary theory: (1) that unisexual species should be
short lived and less speciose than their outcrossing counterparts and (2) that androdioecy (mixtures of
males and hermaphrodites) is an unstable, transitionary system that should not persist over long periods
of time. We nd no evidence of all-unisexual spinicaudatan taxa (clam shrimp) in the fossil record, but
do nd evidence of both androdioecious and dioecious clam shrimp. We nd that clades with many
androdioecious species are less speciose but persist longer than their mostly dioecious counterparts.
These data suggest that all-unisexual lineages likely do not persist long whereas mixtures of unisexual
and sexual breeding can persist for evolutionarily long periods but tend to produce fewer species than
mostly sexual breeding.
Key words: Evolution of sex, Sexual dimorphism, Morphometrics, Androdioecy, Chonchostraca.
Citation: Astrop TI, Park Boush L, Weeks SC. 2020. Testing Weissman’s lineage selection model for the maintenance of sex: the evolutionary
dynamics of clam shrimp reproduction over geologic time. Zool Stud 59:34. doi:10.6620/ZS.2020.59-34.
BACKGROUND
The functional signicance of outcrossing sexual
reproduction has intrigued biologists from the very
inception of evolutionary biology. Darwin (1859) mused
that organisms that refrained from outcrossing sexual
reproduction would “diminish vigour and fertility” and
that “no organic being self-fertilises itself for an eternity
of generations; but that a cross with another individual
is occasionally—perhaps at very long intervals—
indispensable.” Indeed, Weismann (1889) elaborated
on this idea by suggesting that “all species with purely
parthenogenetic reproduction are sure to die out; not,
indeed, because of any failure in meeting the existing
Zoological Studies 59:34 (2020)
doi:10.6620/ZS.2020.59-34
1
© 2020 Academia Sinica, Taiwan
conditions of life, but because they are incapable of
transforming themselves into new species, or, in fact, of
adapting themselves to any new conditions.” The notion
that outcrossing sexual reproduction allowed species to
persist in the face of changing environments persisted
for more than a century before being challenged as
being a “group selection” argument by Williams
(1966) in his seminal book. Since then, a plethora of
“individual selection” hypotheses have been generated
to replace Weismann’s hypothesis (Williams 1975;
Bell 1982; Kondrashov 1993), most of which have not
been successfully borne out (Otto 2009). The lack of
denitive support for short-term benets to outcrossing
sexual reproduction has logically led to theoretical re-
examination of Weissman’s ideas (termed the “lineage
selection” model) as a mechanism to maintain sexual
reproduction (Nunney 1989; Burt 2000; de Vienne et al.
2013).
Even though the notion that unisexual lineages
should be evolutionarily short-lived and less speciose
than their outcrossing sexual counterparts (Weismann
1889; Fisher 1930; Muller 1932 1964) is indeed a
“group selection” hypothesis, it may nevertheless either
partially or wholly explain the predominance of sexual
reproduction in the plant and animal kingdoms (Nunney
1989 1999; Burt 2000; de Vienne et al. 2013). Because
of the long time frames dictated by the lineage selection
model, empirical tests of this hypothesis have only
been indirect; the scarcity of major clades of wholly
or predominantly unisexual lineages—for example
ostracods (Schön et al. 2009), oribatid mites (Norton
et al. 1988), spinicaudatan ‘clam shrimp’(Weeks et al.
2009) and bdelloid rotifers (Arkhipova and Meselson
2000; Welch et al. 2004; Fontaneto et al. 2007 2012)—
has been cited as indicative of the lineage-selection
model (Bell 1982).
In order to test such a temporally-dependent
hypothesis, we would need a readily fossilized clade
that is reproductively labile and from which breeding
system type can be assessed. Unfortunately, to date,
determination of reproductive mode of fossils has
been problematic, and in those taxa that show sexual
dimorphism [e.g., ammonites (Longridge et al. 2008;
Zatoń 2008), ostracods (Ozawa 2013), and vertebrates
(Klein et al. 2012)], reproductive mechanisms are often
invariant, disallowing empirical comparison. Because
of this, palaeontological tests of the long-term benets
of sexual reproduction in multicellular organisms
(Weismann 1889; Fisher 1930; Muller 1932 1964) have
been impossible.
There is one clade—branchiopod crustaceans in
the suborder Spinicaudata (Fig. 1)—that does fit the
above criteria. These clam shrimp exhibit a diversity
of reproductive systems: dioecy (males + females),
androdioecy (males + hermaphrodites) and selfing
hermaphroditism (Sassaman 1995; Brantner et al. 2013;
Weeks et al. 2014). Additionally, unisexuality (i.e.,
selfing hermaphroditism) has independently evolved
a minimum of four times from dioecious ancestors
(Weeks et al. 2014). These crustaceans readily fossilize,
and have a rich fossil record that is well established
(Raymond 1946; Novojilov and Kapeljka 1960; Tasch
and Shaffer 1964; Zhang et al. 1976; Tasch 1987;
Gallego and Martins-Neto 2006; Kozur and Weems
2007; Astrop and Hegna 2015; Hethke et al. 2019).
Recent methodological breakthroughs (Astrop et al.
Fig. 1. The limnadiid spinicaudatan Calalimnadia mahei. b: Brood chamber with eggs, h: Head, p: Phyllopodous thoracic limbs, t: Telson.
page 2 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
2012) have allowed the extraction of sex ratio estimates
from fossil clam shrimp to ascertain reproductive
systems in fossil populations of Spinicaudata (Monferran
et al. 2013; Stigall et al. 2014). The combination of
these factors allows us a unique opportunity to test
Weissman’s original hypothesis: that unisexual species
should be short lived and less speciose than their
outcrossing counterparts (Nunney 1989; de Vienne et al.
2013).
Herein, we use Astrop et al.’s (2012) shape
comparison methodology to assess sex ratios in fossil
clam shrimp allowing us to assign mating systems
to fossil species in a reproductively diverse taxon:
the Spindicaudata (Weeks et al. 2008). We then use
these analyses to directly address Weissman’s (1889)
original predictions that unisexual species should be
less speciose and shorter-lived than their dioecious
counterparts, as well as to assess the prediction that
mixtures of males and self-compatible hermaphrodites
(androdioecy) should be short-lived (Charlesworth
1984).
MATERIALS AND METHODS
Sampling for this study was conducted at multiple
museums and repositories across the world (Tables 1
and 2). Specimens were processed using a ‘portable
imaging station’ which comprised a Nikon D3000,
macro-lens, tripod, lighting, laptop computer and image
capture/editing software.
Morphometric Protocol
The outlines of individual carapaces were
digitized using tpsDig v2.10 (Rohlf 2006) and then
subjected to standard eigenshape analysis. The protocol
and proof of concept utilized in this study is covered in
depth in Astrop et al. (2012). A brief description of the
methodology follows.
Eigenshape analyses (sensu MacLeod 1999)
operate via the conversion of the digitized outline of
an individual specimen into equidistant, Cartesian (x-
y) coordinates. These coordinates are subjected to a
generalized Procrustes analysis (GPA sensu Bookstein
1996 1997) in order to remove the effect of size,
location and rotation and allow the data to projected
into a two dimensional space. The Procrustes-aligned
coordinates are then transformed into a shape function
as angular deviations (phi function: φ; Zahn and Roskies
1972) from the previous step (coordinate) in order
to describe the shape of the curve. This description
is derived from a set of empirical, orthogonal shape
functions via an eigenfunction analysis of a matrix of
correlations between shapes. Eigenshape ‘scores’ can be
then used to project individual specimens into a multi-
dimensional morphospace that allows the visualization
of individual vectors of shape change and highlight
whether particular vectors of deviation from the ‘mean
shape’ are characteristic of a particular group.
Digitized outline data was then processed using
modified versions of the Eigenshape v2.6 & Guide to
Models v0.7 Mathematica notebooks available via the
morphotools site (http://www.morpho-tools.net). The
analysis interpolates and standardizes the raw Cartesian
data before performing a singular value decomposition
to produce eigenvalues, eigenscores and eigenshapes
that describe variation of shape within the dataset.
Size is removed from the analysis as eigenshape axis
one which is manually discarded and the second
eigenshape reported by the analysis is treated as the
‘true’ first eigenshape (ES1) describing shape change.
The eigenshapes produced by the analysis describe
two-dimensional axes of shape change that can be
Table 1. Number of fossil species represented by adequate numbers to be of use in this
study from visited institutions
Collection # of viable species
CONICET 7
NHM 5
SMNH 8
PIN 2
NIGPAS 4
AMNH 3
Institution abbreviations: CONICET, National Research Council Scientic and Technical, Corrientes, Argentina.
NHM, Natural History Museum, London, UK. SMNH, Smithsonian Museum of Natural History, DC, USA. PIN,
Paleontological Institute, Russian Academy of Sciences, Moscow. NIGPAS, Nanjing Institute of Geology and
Palaeontology Chinese Academy of Sciences, China. AMNH, Australian Museum of Natural History, Sydney,
Australia.
page 3 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
used to construct morphospaces that specimens may
be projected onto, allowing trends in shape variation
to be observed. The eigenscores can then be used in
a simple cluster analysis to evaluate the existence of
morphotypes that should correspond to sex.
The current study employed some changes to the
protocol outlined in Astrop et al. (2012). Astrop (2014)
found that using 10 rather than 500 equidistant points
reduced the likelihood that taphonomic and/or human
error would enter the analysis while extracting the same
level of useful shape information from the carapaces
of extant Spinicaudata. Thus, we used this less “noisy”
method herein.
Evolutionary Context
In order to provide a framework for interpreting
the evolutionary dynamics of sexual systems over
geologic time, hypothesized relationships between
extinct taxa were based on Zhang et al. (1976). Trees
were constructed manually in Mesquite (V2.75) based
on existing literature (e.g., Novojilov 1961; Zhang et
al. 1976; Chen and Hudson 1991) to produce les in a
nexus format that were manageable by the R language
environment and associated phylotools package (Revell
2012).
Unfortunately, most phylogenetic methods and
reconstructions do not take into account terminal taxa
becoming extinct before the present or the sampling
error intrinsic to palaeontological data. Thus, in these
analyses, the R package paleotree (Bapst 2012) was
utilized, which allowed for time-scaling of branches in
the tree and testing for serious issues in assuming the
data collected are representative of the actual diversity
of the fossil group.
Statistical tests regarding the distribution and
duration of sexual systems in fossil groups were
performed in R and PAST (Hammer et al. 2001)
RESULTS
Monomorphism vs. Dimorphism
In order to establish the presence of different
reproductive phenotypes, we must rst establish that the
methods outlined in Astrop et al. (2012) can eectively
discriminate monomorphism (i.e., parthenogenesis
Table 2. Metadata of fossil material used in this study. Institution abbreviations same as in table 1
Species Familial aliation Collection Specimen # Age Useful Eigen-
shapes
% variance
captured
Carapacestheria
disgregaris
Eosestheriidae (Shen 1994) NHM London, Ohio
University (OU), SMNH
NHM it2566-81 Jurassic 1,2 72%
Martinestheria
(Lioestheria) codoensis
Antronestheriidae (Gallego et
al. 2013)
Argentina Uncurated Lower Cretaceous 1,2,3 49%
Challaolimnadiopsis
mendozaensis
Eosestheriidae (Sensu Zhang et
al., 1976)1
Argentina Uncurated Triassic 1,2,3 72%
Wolfestheria smekali Fushunograptidae (Wang) in
Hong et al. 1974
Argentina Uncurated Upper Jurassic 1,2,3 36%
Menucoestheria
wichmanni
Eosestheriidae (Zhang et al.,
1976)
Argentina Uncurated lower Upper Triassic 2,3 20%
Leaia gondwanella Leaiidae (Raymond 1946) SMNH usnm426155 Mid-Upper Permian 1,2 34%
Estheria forbesi (all) Eosestheriidae (Sensu Zhang et
al., 1976)1
NHM London, Argentina NHM in44340 - 51883, ARG
“New stu in tissue”, TA1-TA7
Triassic 1,2 59%
Cyzicus (Euestheria)
crustapatulis
Euestheriidae1SMNH usnm427800/06 & usnm427807 Lower Jurassic 1,2 30%
Eosolimnadiopsis
santacrucensis
Eosestheriidae (Sensu Zhang et
al., 1976)1
Argentina Uncurated Jurassic 1,2 67%
Lioestheria
malacaraensis
Fushunograptidae (Gallego et
al. 2011)
Argentina, SMNH usnm427989 Jurassic 1,2,3,4 90%
Euestheria taschi Euestheriidae (Monferran et
al., 2013)
Argentina 5718 middle Late-Jurassic 1,2,3,4 91%
Estheria mangliensis Euestheriidae1NHM London NHM in4961 - 35274 Upper-Triassic 1,2 68%
Euestheria mangliensis
(?)
Euestheriidae1Argentina Uncurated middle Late-Jurassic 1,2 71%
Estheria mangaliensis Euestheriidae1SMNH Uncurated Upper-Triassic 1,2 54%
Triassoglypta sp. 3 Loxomegaglyptidae (Novojilov
1958)
Argentina Uncurated Late Triassic 1,2,3 82%
Estheria middendor Euestheriidae1NHM London NHM in9262 - un-cataloged Upper Cretaceous 1,2 62%
Leaia leidyi Leaiidae (Raymond 1946) NHM London NHM in3088-3114 Lower Carboniferous 1,2 61%
page 4 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
Species Familial aliation Collection Specimen # Age Useful Eigen-
shapes
% variance
captured
Cyzicus (Euestheria)
formavariabalis
Eosestheriidae (Sensu Zhang et
al., 1976)1
SMNH usnm426198 Lower Jurassic 1,2 68%
Cyzicus (Euestheria)
crustabundis
Eosestheriidae (Sensu Zhang et
al., 1976)1
SMNH usnm427901/4+985 Lower Jurassic 1,2 59%
Cyzicus (Lioestheria)
antarctis
Eosestheriidae (Sensu Zhang et
al., 1976)1
SMNH usnm426177 Lower Jurassic 1,2 58%
Perilimnadia sp. Perilimnadiidae (Sensu Zhang
et al., 1976)1
AMNH Tray L34-CO1 Upper Permian 1,2 59%
Hemicyclolaeia mitchelli/
discoidea
Leaiidae (Raymond 1946) AMNH Tray L34-CO3 Upper Permian 1,2 62%
Cyzicus (Lioestheria)
branchocarus
Euestheriidae1AMNH Tray L34-CO5 Cretaceous 1,2 58%
Estheria simoni Euestheriidae1PIN Uncurated Upper Carboniferous 1,2 73%
Limnadia volgaica Palaeolimnadiidae (Sensu
Tasch 1956)1
PIN 2141/1 Upper Permian 1,2 61%
Eosestheria luanpingensis Eosestheriidae (Zhang et al.,
1976)
NIGPAS 97438-57 Early Cretaceous 1,2 76%
Neodiestheria
changmaensis
Diestheriidae (Chen) in Zhang
et al. 1976
NIGPAS 45564-45566 Early Cretaceous 1,2,3 71%
Dictyestheria elongata/
ovata
Halysestheriidae (Zhang et al.,
1976)
NIGPAS Uncurated Upper Cretaceous 1,2,3 69%
Halysestheria yui Halysestheriidae (Zhang et al.,
1976)
NIGPAS Uncurated Upper Cretaceous 1,2 63%
Species Tot N N = M1 %M1 N = M2 %M2 Predicted sexual system2
Carapacestheria disgregaris 34 17 50.0 17 50.0 D
Martinestheria (Lioestheria) codoensis 15 7 46.7 8 53.3 D
Challaolimnadiopsis mendozaensis 14 7 50.0 7 50.0 D
Wolfestheria smekali 33 14 42.4 19 57.6 D
Menucoestheria wichmanni 23 9 39.1 14 60.9 D
Leaia gondwanella 16 6 37.5 10 62.5 D
Estheria forbesi (all) 116 63 54.3 54 46.6 D
Cyzicus (Euestheria) crustapatulis 33 10 30.3 23 69.7 A
Eosolimnadiopsis santacrucensis 48 17 35.4 31 64.6 A
Lioestheria malacaraensis 55 38 69.1 17 30.9 A
Euestheria taschi 20 9 45.0 11 55.0 D
Estheria mangliensis 38 13 34.2 25 65.8 A
Euestheria mangliensis (?) 38 13 34.2 25 65.8 A
Estheria mangaliensis 61 33 54.1 28 45.9 D
Triassoglypta sp. 3 28 11 39.3 17 60.7 D
Estheria middendor 34 11 32.4 23 67.6 A
Leaia leidyi 30 10 33.3 20 66.7 A
Cyzicus (Euestheria) formavariabalis 13 / / / / N/A
Cyzicus (Euestheria) crustabundis 17 / / / / N/A
Cyzicus (Lioestheria) antarctis 17 8 47.1 9 D
Perilimnadia sp. 28 / / / / N/A
Hemicyclolaeia mitchelli/discoidea 45 12 26.7 33 73.3 A
Cyzicus (Lioestheria) branchocarus 41 21 51.2 20 48.8 D
Estheria simoni 17 7 41.2 10 58.8 D
Limnadia volgaica 19 9 47.4 10 52.6 D
Eosestheria luanpingensis 37 17 20 54.1 D
Neodiestheria changmaensis 60 28 46.7 32 53.3 D
Dictyestheria elongata/ovata 99 N/A
Halysestheria yui 69 33 47.8 36 52.2 D
1suggested change. 2A = androdioecy; D = dioecy.
Table 2. (Continued)
page 5 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
or all hermaphrodites) from dimorphism (dioecy or
androdioecy). Thus, we began this study by subjecting
known sexes of differing combinations to analysis:
a sample of 15 males and 15 hermaphrodites of the
extant androdioecious Eulimnadia texana Packard,
1871 was used as the “dimorphic” population and 15
hermaphrodites as the “monomorphic” population.
The first two eigenshape axes contained 82% of
the variation for the dimorphic dataset and 57% of
the variation observed in the monomorphic dataset.
Hierarchical cluster analyses of these respective datasets
(Fig. 2) revealed two groups separated by long branch
lengths (relative to disparity between either cluster’s
eigenshape scores) in the dimorphic dataset (Fig. 2B)
whereas in the monomorphic dataset (Fig. 2A) branch
lengths were considerably lower and did not show the
distinct grouping seen in the dimorphic dataset. Thus,
the method implemented by Astrop et al. (2012) can
successfully distinguish between a monomorphic vs. a
dimorphic data set.
Fossil Comparisons
A total of 29 species of fossil Spinicaudata
represented by between 30–200 individuals were used in
these analyses (Tables 1–3). Individual fossil specimens
from collections were deemed viable if there was little
to no visible taphonomic interference in the outline
of the preserved carapace valve (approximately 30%
of observed specimens were of suitable preservation).
Small shape variations in individual specimens are
described by the eigenshape analysis as ‘non-ane’ or
non-uniform. This non-uniform variation is likely to be
relegated to lower eigenshapes as ‘noise’ whereas more
uniform or ‘ane’ shape change, that is, trends in shape
change seen across specimens in the dataset, comprised
the majority of variance captured by higher eigenshapes.
A total of 1,098 specimens from 29 species (Table 2)
were analyzed using the morphometric protocol outlined
above.
Observing the branching patterns in the extant (Fig.
2) examples and fossil examples (Fig. 3), similarities
and differences are clear. The fossil taxon Lioestheria
malacaraensis Tasch 1987 (Fig. 3B) definitively
displays a strong basal dichotomy in shape variation of
a magnitude similar to that seen in the dimorphic dataset
of the extant Eulimnadia texana (Fig. 2B). Conversely
Palaeolimnadia sp. (Fig. 3A) exhibits no clear clusters,
reminiscent of the monomorphic dataset of E. texana
(Fig. 2A). A major difference between the patterns
seen in Palaeolimnadia sp. versus that seen in the
monomorphic E. texana data is the size of the Euclidean
distance between specimens. This distance measure
is an effective way of discerning groups because data
contained in the vectors are all in the same physical units
(a measure of disparity in shape, with size, scaling and
rotation removed). The distance between specimens in
the Palaeolimnadia sp. dataset (Fig. 3A) is of an order
of magnitude higher than that seen in the monomorphic
E. texana dataset (Fig. 2A) and is very similar to
distance measures in other dimorphic taxa studied
(Astrop et al. 2012). This can be simply interpreted
as there being very little difference in shape between
individuals in the monomorphic E. texana data set and
dierences in shape between multiple individuals in the
Palaeolimnadia sp. data set of a magnitude similar to
Fig. 2. Cluster analyses of a monomorphic (hermaphrodites only) sample of Eulimnadia texana (A) and a dimorphic (males + hermaphrodites)
sample (B) based on scores of individuals along the rst four eigenshape axes. Note the diering distances along the Y-axes in the two graphs.
page 6 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
that seen in the dimorphic data. Therefore, we interpret
these patterns in Palaeolimnadia sp. (Fig. 3A) as
either reective of the presence of multiple species in a
collection labeled as a single species, or as being caused
by severe taphonomic interference in these specimens.
We found these patterns in four of the 29 (~14%) taxa
examined: Dictyestheria elongata / D. ovata Chang and
Chen 1963, Palaeolimnadia sp., Cyzicus (Euestheria)
Table 3. Fossil clam shrimp measured
Name Reference
Carapacestheria disgregaris Tasch 1987
Challaolimnadiopsis mendozaensis Shen et al. 2001
Cyzicus (Euestheria) crustabundis Tasch 1987
Cyzicus (Euestheria) crustapatulis Tasch 1987
Cyzicus (Euestheria) formavariabalis Tasch 1987
Cyzicus (Lioestheria) antarctis Tasch 1987
Cyzicus (Lioestheria) branchocarus Talent 1965
Dictyestheria elongata/ovato Chang and Chen 1964
Euestheria luanpingensis Zhang et al. 1990
Eosolimnadiopsis santacrucensis Gallego 1994
Estheria forbesi all Jones 1862
Estheria mangaliensis L3 Jones 1862
Estheria mangliensis L1 Jones 1862
Estheria middendor Jones 1862
Estheria simoni Pruvost 1911
Euestheria mangliensis L2 Jones 1862
Euestheria taschi Vallati 1986
Halysestheria yui Chang 1957
Hemicylcolaeia discoidea/mitchelli Mitchell 1925; Etheridge 1892
Leaia gondwanella Tasch 1987
Leaia leidyi Lea 1855
Limnadia volgaica Novojilov 1970
Lioestheria malacaraensis Tasch 1987
Martinsestheria codoensis Cardoso 1962
Menucoestheria wichmanni Gallego 2010
Neodiestheria changmaensis Shen and Chen 1982
Paleolimnadia sp. Tasch and Oesterlen 1977
Triassoglypta sp. 3 Gallego 2005
Wolfestheria smekali Mongerran et al. 2013
Fig. 3. A, Cluster analysis of Palaeolimnadia sp. based on informative eigenshapes (ES1 & 2); no discernible major groupings and multiple long
branches of similar length interfere with deducing sexual system based on a clear morphotype ratio. B, Cluster analysis of Lioestheria malacaraensis
based on informative eigenshapes (ES1 & 2) exhibiting a clear basal dichotomy with branch lengths much larger than any subsequent groupings.
page 7 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
formavariabalis Tasch 1987 and Cyzicus (Euestheria)
crustabundis Tasch 1987. Thus, for these taxa, no sexual
system could be inferred.
For the remaining taxa, morphotypes were
assigned when cluster analysis of the informative
eigenshape scores either produced two distinct groups
(such as in Figs. 2B and 3B) or a single grouping (such
as in Fig. 2A). We found that none of the remaining 25
taxa showed a pattern indicative of a single sex (i.e.,
as in Fig. 2A). Instead, all 25 taxa had two distinct
groupings separated by large Euclidian distances, as
seen in gures 2B and 3B. Thus, these analyses resulted
in two sexes being present among the samples of each
of these 25 taxa.
Actual sexes may be assumed for the two distinct
morphotypes in two ways. First, although naturally
occurring extant populations often display fluctuating
frequencies of sexes (often seasonally), the average
ratio over the season remains indicative of the sexual
system employed by the species (50:50—dioecious;
30:70 male: “female”—androdioecious). This has
been recorded in extant, wild populations of dioecious
species such as Cyzicus tetracerus (Krynicki 1830)
(Popović and Gottstein-Matočec 2006), Leptestheria
nobilis Sars, 1900 (Karande and Inamdar 1959) and the
androdioecious species E. texana (Strenth 1977). When
considering ecological observations, alongside the fact
that fossil-bearing strata usually represent multiple
generations living and dying over time, obtaining
morphotype (or ‘sex’) ratios for a fossil taxon from
such a deposit should reect the ratio representative of
the sexual system of that taxon. Second, rare instances
of soft part preservation have allowed the matching
of claspers (male copulatory appendages) to specific
carapace shapes; for instance, soft part preservation in
the Jurassic euestheriid Euestheria luanpingensis (Zhang
et al. 1990), where claspers are associated with more
elongated sub-quadrate carapace shapes and eggs are
preserved within sub-spherical carapaces.
Fossil taxa were interpreted as displaying a sex
ratio indicative of androdioecy if one morphotype
comprised less than 35% of the sample. This percentage
was used because it is close to the 30/70 ratio (male/
female respectively) that is observed in most extant
androdioecious species (Weeks et al. 2008) but
leaves some room for sampling error. Examples of
morphotypes in fossil species can be seen in gure 4.
Seven of the 25 fossil taxa included in this
analysis exhibited distinct morphotypes with a skewed
frequency where the less common morphotype made
up 35% of the sample or less. These taxa occurred
in three of the nine families studied (Fig. 5): the
Leaiidae, Fushunograptidae and Euestheriidae. Two
of three taxa in the Leadiidae, one of two taxa in
the Fushunograptidae and four of eight taxa in the
Euestheriidae exhibited androdioecious sex ratios.
Interestingly, where androdioecy was suggested in a
fossil family, it seemed to occur in at least half of the
species sampled in that family (Fig. 5).
Polytomies in the tree presented in figure 5
originate from uncertain intra-familial relationships
inferred by Zhang et al. (1976) and revised by Astrop
and Hegna (2015). However, by time-calibrating the
tree using the software package Paleotree (Bapst 2012),
it was possible to bound rst occurrences in the fossil
record to branches and resolve polytomies according
to (in this case) the range of geologic stages through
which the genera occur (Fig. 6). This revised analysis
adds information that would otherwise be lost and that
is often ignored in modern phylogenetic studies that
incorporate extinct taxa. Time-scaling the tree shows
that androdioecious lineages have occurred multiple
times since the Devonian. The durations of the branches
(Fig. 6) are reflective of the first and last known
occurrences of species within that genus in the fossil
record.
It is clear that the fossil clam shrimp have two
distinct clades both originating ~300 mya (Fig. 6). In
this case, clade A has 15 species and only a single case
of a skewed sex ratio (Lioestheria malacaraensis).
The breeding system of three of the species in this
clade were not determined (Dictyestheria elongata / D.
ovata, Cyzicus (Eustheria) formavariabalis and Cyzicus
(Eustheria) crustabundis), but the remaining 11 species
were determined to be dioecious. Thus, the range of
dioecy possible for this clade is ~73–93% dioecious.
Clade A is determined to be a primarily dioecious (PD)
clade. Clade B has only seven species, of which four are
androdioecious. Thus, this clade is 57% androdioecious.
Clade B is considered the primarily androdioecious
(PA) clade. We can compare the PD to the PA clade
in two ways: number of species per clade and average
species duration in the fossil record. For the former, we
used a binomial test to assess the likelihood of equal
numbers of species being distributed in the two clades
because the clades appear to be approximately equally
old (300–320 my). The likelihood that the two clades
are actually equally speciose is 0.041, and thus the PD
clade has signicantly more species than the PA clade.
Using the species duration estimates from gure 6, we
found that the PA average duration (57.4 ± 8.7 MY) was
signicantly longer (F1,20 = 11.45; P < 0.0030) than the
PD average duration (21.9 ± 5.9 MY) indicating that the
PD clade has more, shorter-lived species than the PA
clade (Fig. 6).
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© 2020 Academia Sinica, Taiwan
DISCUSSION
Although the theory that sexual reproduction has
been maintained because it allows organisms to adapt
to evolutionarily rapid changes in the environment
(Weismann 1889) has been largely discounted because
it was determined to be “group-selectionist” (Williams
1966; Maynard Smith 1971 1978), an eective empirical
test of this theory in metazoans has been impossible
until now. Herein, we have made the first such test,
using fossil species from the reproductively labile clam
shrimp as our study organism. From these comparisons,
we have made two important discoveries: (1) all-female/
hermaphrodite species were not observed in the fossil
data studied (2) there were several fossil lineages with
sex ratios indicative of androdioecy, and those species
were non-randomly divided into two clades that diered
in average species duration and species number.
Fig. 4. A sample of the fossil taxa studied, their diagnosed sexual systems and overlaid mean-shapes of the detected morphotypes (M1 = Morphotype 1;
M2 = Morphotype 2).
page 9 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
Lack of All-Unisexual Fossil Clam Shrimp
Out of the 29 fossil clam shrimp species examined,
we found no instances of genuine monomorphism and
therefore no cases of inferred unisexuality. Although
we were unable to determine the sexual system of four
fossil species, none of these four species had patterns
indicative of a single-sexed species (Fig. 2A). Instead,
these four species had long-branch patterns indicative
of either mixed species assemblages or taphonomic
interference causing carapace outline distortion (see
Fig. 3A for an example). Thus, out of the 25 species of
which we could determine breeding system type, we
found no evidence of all-unisexual species, suggesting
that if purely hermaphroditic taxa occurred in the
Spinicaudatan clade in the geologic past, they are
not represented in the fossil record so far examined
(covering the past 370 million years).
This dearth of all-unisexual species in the fossil
record is mirrored in the extant clam shrimp species
so far studied. Among all of the extant Spinicaudata,
unisexuality (i.e., selng hermaphroditism) has evolved
four independent times (Weeks et al. 2014), with a
fifth independent derivation proposed but not verified
(Roessler 1995). Of the four transitions away from
dioecy, three have been to unisexuality (i.e., species
comprised solely of self-fertilizing hermaphrodites)
and one has been to androdioecy (i.e., ~30% males +
~70% self-fertilizing hermaphrodites). One of the three
unisexual taxa is the monospecic Calalimnadia mahei
Rabet & Rogers, in Rogers et al. (2012). The genus
Limnadia comprises three unisexual species (Bellec et
al. 2018). The third (Cyzicus gynecius (Mattox 1950))
is in a genus of 26 species (Rogers 2020). The fourth
derivation is in the genus Eulimnadia, which appears to
be predominantly androdioecious (Weeks et al. 2009)
and contains about 45 species (Rogers 2020). Overall,
the Spinicaudata are primarily dioecious (Sassaman
1995), containing approximately 200 total species
(Rogers 2020).
The combined evidence of a complete absence
of carapace monomorphism in the fossil taxa in our
study, with the few examples of all-unisexual species
among extant clam shrimp species, supports the
hypothesis that all-unisexual species (in the case of the
Spinicaudata, species that are exclusively self-fertilizing
hermaphrodites) should be prone to extinction and less
speciose than their dioecious counterparts (Weismann
1889; Fisher 1930; Muller 1932 1964).
Because we found no unisexual species in any
of the 25 fossil species surveyed, we can infer that
less than 4% (fewer than one out of 25) of fossil clam
Fig. 5. Distribution of predicted sexual systems in fossil taxa analyzed in this study. NA = taxa in which sexual system prediction was impossible (see
MATERIALS AND METHODS).
page 10 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
shrimp are all-unisexual. Only four of ~200 extant
clam shrimp species (~2%) are all-unisexual, and those
few all-unisexual species tend to be in monomorphic
clades or at the tips of their respective phylogenetic
trees (Weeks et al. 2014). These data suggest that all
unisexual lineages are shorter-lived and less speciose
than their outcrossing counterparts, as originally
suggested by Weismann (1889).
In Weismann’s original formulation of this
hypothesis, unisexual lineages would be evolutionarily
short-lived because of their inability to evolve quickly
enough to keep up with a changing environment
(Weismann 1889; Muller 1932). A more modern twist
on Weismann’s original idea suggests that unisexual
lineages are short lived because of the accumulation of
deleterious alleles (i.e., ‘Muller’s ratchet’; Muller 1964)
and the subsequent ‘mutational meltdown’ that occurs
when population sizes decline due to the eects of this
mutation accumulation (Lynch et al. 1995). This is the
first corroborative evidence of Weismann’s original
idea in a multicellular animal lineage, and suggests that
the longer-term “group selection” forces of dierential
speciation and extinction (Nunney 1989) may select
for outcrossing sexual reproduction within the
Spinicaudata. Clearly, because of the long time frames
of this comparison, we have no direct evidence of “group
selection,” per se. Nonetheless, the patterns shown
among the fossils and mirrored in the extant species are
indicative of low speciation and short duration for all-
unisexual species of Spinicaudata.
Parity of Androdioecious and Dioecious Clam
Shrimp Species
Seven of the 25 fossil species in which breeding
system could be determined where found with sex
ratios indicative of androdioecy, with the remaining 18
species having sex ratios indicative of dioecy (Table
2). The average number of fossil specimens examined
for the eight androdioecious species was ~40 (Table
2), which would yield an expected ~2% chance of
finding a sex ratio of 35% males if the true sex ratio
was 50% males. Because we sampled 25 species,
we would expect less than one of these 25 species to
be mistakenly categorized as androdioecious when
it was actually dioecious, given our sex ratio cut-off
and the average sample sizes of those species found
to be androdioecious. Given that we instead found
Fig. 6. A time-calibrated phylogeny with polytomies largely resolved by stratigraphic occurrence and branch lengths approximate to generic range.
X axis = millions of years.
page 11 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
seven species to be androdioecious suggests that the
reproductive lability in extant clam shrimp (Weeks et
al. 2008) is reflective of a general tendency towards
reproductive lability in clam shrimp over geologic time
(Fig. 6). A similar level of lability (also in the form
of androdioecious and dioecious species) has been
described in the closely related branchiopod crustacean
group Notostraca (Mathers et al. 2013), suggesting
that the Branchiopoda have a genetic system that tends
towards the repeated evolution of sperm production in
females."
The causes and mechanisms of this suggested
reproductive lability remain relatively unknown.
However, Weeks et al. (2006) proposed that “females”
with small amounts of sperm production can be
produced via low levels of crossing over between
the sex chromosomes in these crustaceans; these
“intersexes” could then be further modified over
evolutionary time to be increasingly t hermaphrodites.
Such repeated evolution of sperm-producing “females”
can lead to the spread of hermaphrodites, which may
eventually outcompete either (a) females to form
androdioecy or (b) both males and females to produce
unisexual (i.e., selng hermaphrodite-only) species. The
selective pressure for such a hermaphroditic spread is
likely an adaptation to harsh, uctuating environments
where reproductive assurance is beneficial (Pannell
1997 2002). However, the current data suggest that any
lineage that subsequently loses males essentially passes
a ‘Rubicon’ after which it is doomed to extinction. This
‘point of no return’ is likely associated with the mode
in which hermaphroditism occurs in the Spinicaudata:
if the frequency of hermaphroditic individuals becomes
high enough to establish large numbers of monogenic
populations (as monogenic hermaphrodites have the
greatest reproductive assurance, always producing self-
fertilizing hermaphrodites that cannot produce or cross
with males), these populations may eventually out-
reproduce and succeed any amphigenic populations
(consisting of female-biased hermaphrodites that are
able to produce both hermaphrodite and male ospring)
maintaining males. This would quickly eliminate
outcrossing from a lineage, ultimately leading to
extinction.
The macroevolutionary patterns of lineage
duration and speciation rates in the primarily dioecious
(PD) and primarily androdioecious (PA) clades did
not conform to expectations. Extant androdioecious
clam shrimp species commonly form all-unisexual
populations (Weeks et al. 2009) and in some cases
whole species are unisexual (Weeks et al. 2005, Bellec
et al. 2018). Thus, extending Weismann’s (1889)
original argument, we expected that PA clades would
produce androdioecious species that were both shorter
lived and less speciose than PD clades. We did indeed
nd that the PD clade was signicantly more speciose
than the PA clade, but the PA clade has species that
survived significantly longer than the average PD
species (Fig. 6). The extant, primarily androdioecious
clade Eulimnadia is also quite long-lived (Weeks et al.
2006), suggesting that androdioecy, per se, does not
doom a lineage to extinction. These combined data also
fail to support the idea that androdioecy is an unstable,
transitionary sexual system (Charlesworth 1984), given
that androdioecious fossil taxa as old as 370 million
years apparently persisted within families for at least 70
million years (Fig. 6).
The pattern seen between PA and PD clades
could have been produced in a number of ways. It
is possible that the PD clade tends to spin off more
species, but many of those species are shorter lived
than in the PA clades. This might suggest that dioecy
allows a more “exploratory” evolutionary trajectory
than androdioecy, but that many of those “experiments”
fail. An alternate explanation is that the androdioecious
breeding system is very stable in these environments
(temporary freshwater pools) and that any evolutionary
“exploration” may spin off exceptionally short-lived
unisexual lineages (i.e., all selng hermaphrodites) that
quickly go extinct. Androdioecy has been suggested as a
mechanism to assure reproduction in habitats with high
population turnover (Pannell 1997 2002). It is possible
that the PA crustacean lineage has historically populated
such habitats and thus that androdioecy has been an
optimal strategy for them for these longer time frames.
Likewise, the PD clade may occupy niches that are
relatively more stable.
We are well aware that the comparative fossil
data among these two clades of clam shrimp are not
strong enough to make broad generalizations, nor to
discern which (if any) of the above explanations may
have caused the patterns we observed. However, these
data allow us to begin to speculate as to causation for
the patterns observed and to address questions that
have heretofore not been addressable in metazoans.
We will need more reproductive data from fossil clades
in reproductively labile groups before we can better
understand the patterns of persistence and speciation
dierences between dioecious and unisexual lineages.
Macroevolutionary Patterns within the
Branchiopoda
The exact pattern of the emergence and
maintenance of androdioecy and dioecy in the
Spinicaudata and Branchiopoda is only beginning to
be explored (Hoeh et al. 2006; Weeks et al. 2009 2014;
Mathers et al. 2013). However, our exploration of the
page 12 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
spinicaudatan fossil record begins to shed light on the
duration, emergence and disappearance of lineages
that exhibit different breeding systems within the
Branchiopoda. Figure 7 represents two possible patterns
of breeding system dynamics in the Branchiopoda based
on the data collected in this study. The rst represents
a scenario where each occurrence of androdioecy
in living and fossil taxa is independent and all arise
from a dioecious ancestor. The second scenario entails
one single occurrence of androdioecy in an ancestral
phyllopod and four subsequent losses in spinicaudatan
clades. Despite the second hypothesis being the most
parsimonious, involving only five state changes
(occurrence/disappearance of sperm production in
females), the rst scenario is not only more biologically
intuitive (given the sex determination deduced for
Spinicaudata in neontological studies) but also is
supported by recent molecular analyses and attempts
at ancestral state reconstruction (Mathers et al. 2013;
Weeks et al. 2014).
The fossil taxa in this study carry a clear
palaeontological signal that suggests the hypothesized
reproductive lability inferred from studies of living
Spinicaudata is ancient, occurring multiple times over
the past 370 million years. It appears that this unique
crustacean clade has been able to use this lability
successfully throughout the geologic past to claim both
the benets of unisexual and sexual reproduction while
avoiding the long term negative eects of engaging in
prolonged periods of selfing. These crustaceans have
clearly been highly successful throughout time (often
bestowed the dubious moniker of “living fossils”), and
it is likely that their reproductive lability has contributed
to this long term success.
CONCLUSIONS
The patterns seen in the Spinicaudata offer the
first empirical observations of multiple reproductive
systems occurring in the fossil record of a single
clade and provides a framework for future integrated
biological and palaeontological studies to elucidate the
evolutionary dynamics of biological phenomena over
geologic time. By integrating palaeontological and
biological approaches, we have recovered definitive
evidence for a hypothesized microevolutionary
phenomenon occurring at a macroevolutionary level.
The absence of fossil monomorphic populations in
the Spinicaudata adds weight to the idea that closed,
unisexual lineages are doomed to extinction through
reduced genetic variability and accumulation/exposure
of deleterious mutations. This study also nds evidence
contradicting the idea that androdioecy is an unstable,
transitionary breeding system, occurring in fossil taxa
as old as 370 million years old and persisting within
families that are at least 70 million years old. The
value of these results would be greatly enhanced with
additional molecular evidence and fossil-calibrated
divergence time estimates to increase the accuracy
of predicted lineage durations. It is our hope that the
integrated, multi-pronged approach to investigating
the evolution of breeding systems in living and fossil
Spinicaudata be utilized for similar investigations of
biological interactions in other fossil taxa.
Fig. 7. Two possible scenarios of the evolutionary dynamics of hermaphroditic lability in Spinicaudata at the family level. Red branches: lineages
with some degree of hermaphroditism present in some taxa. Black branches: lineages devoid of hermaphroditic ‘females’ in all taxa. Blue boxes:
sperm production in females occurring. White boxes: Sperm production lost in hermaphrodites recreating females. † = Extinct group.
page 13 of 16Zoological Studies 59:34 (2020)
© 2020 Academia Sinica, Taiwan
Acknowledgments: We thank Oscar Gallego,
Thomas Hegna, Alexy Kotov and Gang Li. This project
was supported from a grant from the National Science
Foundation (DEB-1210587) to TIA and SCW.
Authors’ contributions: TIA helped design the
experiments, collected and analyzed the data and helped
write the paper. LPB and SCW helped design the
experiments and write the paper.
Availability of data and materials: This manuscript
does not contain any personal data beyond that
belonging to the authors.
Competing interests: TIA, LPB and SCE declare
that they have no conicts of interests.
Consent for publication: No consent needed.
Ethics approval consent to participate: Not
applicable.
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