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Speciation in darkness
Biology Letters draft
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Electronic Supplementary Material
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Speciation in caves: experimental evidence that permanent
darkness promotes reproductive isolation
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Rüdiger Riesch, Martin Plath & Ingo Schlupp
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Additional background information
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Evolution of cave faunas
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One branch of evolutionary biology that traditionally focuses on ecological
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speciation—even though the term is rarely used in this context—is the research
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on speciation of cave faunas [1-3]. Two main hypotheses have been proposed to
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explain the reproductive isolation and subsequent independent evolution of
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troglobites (i.e., obligate cave organisms). The traditional view posits that after
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successful colonization of a cave, surface-dwelling populations become
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geographically disconnected from the subterranean population(s) or even go
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extinct (e.g., due to changing climate), leading to reproductive isolation and
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ultimately allopatric speciation (climatic relict hypothesis [4-6]). Alternatively,
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reproductive isolation could result from ecologically-based divergent natural
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selection even in the absence of physical barriers [7], because individuals in
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caves are predicted to show evolutionary change in order to exploit the new
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habitat type or new food sources (adaptive shift hypothesis [6,8-9]; see also the
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“isolation-by-adaptation” concept introduced by Nosil et al. [10-12]).
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Speciation in darkness
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Intriguingly, several cave fishes co-occur with their closely related surface-
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dwelling relatives within the same river system, and the only obvious ecological
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parameter that apparently prevents gene-flow between divergent ecotypes is the
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presence or absence of light. Beside the Poecilia mexicana system [7,13],
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parapatric contact is seen, e.g., between different cave and surface populations
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of Ancistrus sp. in the São Domingo karst area in Brazil [14-15]. Therefore, one
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obvious question comes to mind: what about the possibility that the absence of
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light in itself could be a strong enough selective force to limit gene flow following
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colonization and initial steps of adaptation to cave life? This question motivated
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our present study, as surprisingly little attention has been focused on this
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question in the past. This is probably due, for the main part, to the necessity to
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conduct rather time-consuming life history studies, which form the backbone of
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our present study. Our present study, for example, required the rearing of N =
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145 individual fish over the course of one year, while ensuring strictly
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standardized feeding and other maintenance procedures (see main text) and
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controlling for the onset of sexual maturity on a daily basis.
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Divergent selection and local adaptation in extremophile P. mexicana
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Poecilia mexicana populations in the Cueva del Azufre system (Tabasco,
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Mexico) are characterized by the simultaneous action of two strong selective
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forces: permanent darkness and hydrogen sulphide (H2S) [13,16-17]. Within a
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small geographical scale of only few kilometres, reproductively isolated
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populations of P. mexicana inhabit environments characterized by all possible
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combinations of these two environmental factors: a toxic cave (Cueva del
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Azufre), a nontoxic cave (Cueva Luna Azufre), a toxic surface stream (El Azufre)
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and a variety of nontoxic surface habitats in the Río Grijalva ⁄ Usumacinta
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drainage (including the two evaluated in our present study: Arroyo Bonita and
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Río Amatan) [13, 16-18]. Toxicity in this system is of volcanic origin [19] and
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reaches concentrations of >300 µM in the Cueva del Azufre [16, 20-21].
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Extremophile P. mexicana are characterized by site-specific local
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adaptations in behavioural (e.g. [22-26]), dietary [27], morphological [17,20-21],
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and physiological traits [28], as well as divergent female [29-32] and male life
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histories [33]. Life-history evolution of extremophile female poeciliids is
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dominated by shifts towards large offspring size and reduced fecundity [29-32],
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while male life history evolution follows a less predictable pattern between
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environments [33]. Nonetheless, population differences in life history traits have
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been found to be largely heritable in males and females [29,31,33].
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Mechanisms of reproductive isolation in the Cueva del Azufre system
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All extreme habitats in the Cueva del Azufre system are interconnected, and no
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physical barriers would prevent individual dispersal and thus gene flow between
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different locally adapted populations [28,34-37]. However, several studies have
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established that populations from ecologically divergent environments are
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reproductively isolated [20,34-36], even after catastrophic flood events that
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temporarily increase the frequency of individual dislocations among habitat types
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[35]. Gene flow at the interface between surface and cave habitats is weak and
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unidirectional, i.e. from the inside of the sulfidic Cueva del Azufre to the sulfidic
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surface habitats but not vice versa [35-36]. What isolating mechanism(s) can
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explain this pronounced genetic structure over extremely small geographic
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distances?
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Previous reciprocal transplant experiments have shown that the presence
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or absence of H2S creates strong reproductive isolation at the interface of toxic to
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nontoxic habitats in natural populations [36,38], while the presence or absence of
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light does not influence 24-hour survival between adjoining toxic cave and
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surface habitats [36]. In addition to H2S-toxicity, predation has been
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demonstrated to contribute to reproductive isolation. First, in controlled
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experiments in the field, giant water bugs (Belostoma sp.) preyed predominantly
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on surface fish in the permanent darkness of the Cueva del Azufre and primarily
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on cave fish in the adjoining surface habitats [39]. Tobler and colleagues [40]
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have also demonstrated that giant water bugs preferentially prey on male P.
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mexicana if given the choice between both sexes. Second, different predator
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regimes between habitats clearly exert additional selection on immigrants. Fish in
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toxic surface habitats suffer from bird predation rates that are more than 20 times
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higher compared to benign surface habitats, but predatory fish are absent from
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extreme habitats [41]. Finally, sexual selection via visual mate choice acts
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against immigrant males in natural populations, because females prefer their own
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males to foreign males [36]. However, previous experiments have shown that,
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while cave-dwelling P. mexicana retained a visual preference for their own
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species [42] and males from their own population [36], they are unable to
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distinguish between ‘own’ and foreign in darkness ([42]; Riesch, Plath & Schlupp,
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unpubl. data). Hence, reproductive isolation as a result of selection from
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predation and sexual selection (at least in light) affects males disproportionately
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more than females.
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The pattern that emerges is quite complex, and is clearly the result of
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multiple isolating mechanisms. The combination of different predation regimes,
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sexual selection via visual mate choice, and hydrogen sulphide toxicity [36,41]
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can clearly explain the lack of gene flow between adjoining toxic and benign
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surface habitats [20,34-36]. However, the mechanisms revealed so far certainly
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fail to fully explain the lack of gene flow from either the toxic surface into the toxic
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Cueva del Azufre, or the nontoxic surface into the nontoxic Cueva Luna Azufre.
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Even more surprising is the fact that according to our previous studies, selection
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should be stronger on cave fish migrating out of the cave, where they encounter
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new predators (i.e., birds [41]) and are selected against via mate choice [36]; yet
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gene flow patterns tell us the opposite story, because we only found weak gene
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flow out of the caves rather than weak gene flow into the caves [20,34-36].
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Potentially, permanent darkness could play an important role as an additional –
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as yet unrecognized – isolating mechanism in this system, and based on the
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known gene flow patterns [20,34-36], we predicted to find decreased fitness of
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surface fish in darkness but not vice versa.
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Additional information on the experimental design
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Common garden protocol
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Field-caught fish were housed in several mixed-sex tanks, in which they were
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exposed to identical environmental conditions (natural light cycle, and no
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hydrogen sulphide or predators). Pregnant females showing a distended
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abdomen were isolated in individual 10-L aquaria, fed ad libitum amounts of
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commercially available flake food, and checked twice daily for offspring until they
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had given birth. Only one clutch per female was included.
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Females were removed from their tanks on the day of birth and measured
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for standard length (SL) to the nearest millimetre. Babies were raised together at
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a maximum density of five babies per 10-L tank for 37 days under ad-libitum
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food- (brine shrimp and ground-up flake food) and benign water-conditions
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(partial water changes were performed every second day). Large clutches (≥10
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babies) were separated into two groups of five offspring per tank; surplus babies
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were discarded from the experiment and introduced into our stock tanks. In total,
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we thus raised N = 44 clutches (11 from AB, 11 RA, and 22 PSV).
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After 37 days, we randomly selected up to four offspring from each clutch
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[i.e., cave mollies often gave birth to less than four offspring [30], so in this case
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we selected all offspring available from that particular clutch up to a maximum of
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four] and randomly assigned them to one of four treatments, leading to an overall
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sample size of 145 individuals in the experiment. Generally, our common garden
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experiment followed well-established protocols (e.g., [43-44]), but some changes
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were made to investigate specific aspects of the cave molly complex: Treatments
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1 and 2 involved a 12:12 hr light/dark cycle coupled with low (tr. 1) or high food
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availability (tr. 2). In treatments 3 and 4, fish were raised in perpetual darkness,
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yet again under low (tr. 3) or high food (tr. 4). Placement of each fish within the
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laboratory setup was also random, but fish from the same clutch were never
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placed upon the same shelf. Feeding regimes followed established protocols
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(e.g., [43-44]), were slightly adjusted to fit mollies according to experience during
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trial runs (R. Riesch, unpubl. data), and were increased every two weeks. For
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example, the original protocols [43-44] were based on feeding measured
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amounts of liver paste in the morning; however, our preliminary studies showed
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that mollies would not grow well on liver paste, so we exchanged liver paste for
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Daphnia. Hence, fish were fed twice daily with a Hamilton micropipette:
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measured amounts of newly hatched Artemia nauplii in the morning, and
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Daphnia in the evening. Since Artemia and Daphnia immediately spread
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throughout the water column of the aquaria, removal of uneaten food remains as
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described in the original protocols [43-44] was not possible. Fish were kept in
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their respective treatments until they were (a) sexually mature and reproductively
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active, or (b) 1 year of age without successful reproduction, at which point they
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were classified as having failed to reproduce.
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No method has been established to visually determine the sex of
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immature mollies (unlike guppies, P. reticulata [45]). Upon entering the
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maturation process, males undergo morphogenetic changes of their anal fin that
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transforms into an intromittent organ, the gonopodium (e.g., [46-48]). Hence, for
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those individuals that eventually turned into males, the experiment was ended
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when anal fin metamorphosis was complete. In the case of females, there are no
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obvious outward signs of sexual maturity, so putative females were mated once a
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week with a mature male of their population from our stock tanks as soon as they
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met one of the following two criteria: (1) one of their siblings from the same clutch
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had successfully undergone metamorphosis to become a mature male, or (2) the
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potential female reached a size of 24 mm, as previous field studies have shown
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that the minimum size of reproducing wild-caught P. mexicana females is around
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28 mm [30]. Females were therefore only scored as ‘reproductively active’, if they
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successfully produced a clutch of offspring within their first year of life.
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