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Wasage Divers, Wakatobi and Buton, Indonesia
Muslimin Kaimuddin

Western Australian Fisheries and Marine Research Laboratories, Department of Primary Industries and Regional Development, Government of Western Australia, North Beach, Western Australia, Australia
Stephen J. Newman & Michael J. Travers

Island Conservation Society Seychelles, Victoria, Mahé, Seychelles
Josep Nogués

CORDIO East Africa, Mombasa, Kenya
Clay Obota & Melita Samoilys

The Centre for Ocean Research and Education, Gregory Town, Eleuthera, Bahamas
Owen O’Shea

Department of Environment and Geography, University of York, York, UK
Kennedy Osuka

Center for Fisheries Research, Ministry for Marine Affairs and Fisheries, Jakarta Utara, Indonesia
Andhika Prasetyo

Universitas Dayanu Ikhsanuddin Bau-Bau, Bau-Bau, Indonesia
L. M. Sjamsul Quamar

Pristine Seas, National Geographic Society, Washington, DC, USA
Enric Sala

Department of Zoology, University of Oxford, Oxford, UK
Melita Samoilys

HJR Reefscaping, Boquerón, Puerto Rico
Michelle Schärer-Umpierre

SalvageBlue, Kingstown, Saint Vincent and the Grenadines
Nikola Simpson

School of Natural and Computational Sciences, Massey University, Auckland, New Zealand
Adam N. H. Smith

Indo Ocean Project, PT Nomads Diving Bali, Nusa Penida, Indonesia
Lauren Sparks

Manchester Metropolitan University, Manchester, UK
Akshay Tanna & Alexandra M. Watts

Reef Check Dominican Republic, Santo Domingo, Dominican Republic
Rubén Torres

Institut de Recherche pour le Développement, UMR ENTROPIE (IRD-UR-UNC-CNRS-IFREMER), Nouméa, New Caledonia
Laurent Vigliola

Secretariat of the Pacific Regional, Environment Programme, Apia, Samoa
Juney Ward

Department of Life Science, Tunghai University, Taichung, Taiwan
Colin Wen

School of Environmental and Forest Sciences, University of Washington, Seattle, WA, USA
Aaron J. Wirsing

Corales del Rosario and San Bernardo National Natural Park, GIBEAM Research Group, Universidad del Sinú, Cartagena, Colombia
Esteban Zarza-Gonzâlez

D. Chapman and M. Heithaus conceived the study with assistance from M. Heupel, C.A.S., M.M., E.H. and M.A.M. D. Chapman, M. Heithaus, M. Heupel, C.A.S., M.M. and E.H. directed fieldwork run by J.G., J.K., M.E.B., L.M.C.-R., C.W.S., K.I.F., J.V.-A., G.C. and C.S.S. Database management was by T. Gorham. M.A.M. and D. Chapman drafted the manuscript, with help from M. Heithaus, M. Heupel, C.A.S., J.E.M.C., M.M., E.H., J.G., J.K., M.E.B., L.M.C.-R., C.W.S., C.S.S., M.J.R., V.U. and T. Gorham. All other authors contributed equally, made substantial contributions to data collection, provided input and approved the text in the manuscript. More

Biodiversity is in crisis and the main reasons are human activities inducing habitat modifications and the introduction of invasive species1. In addition, global climate change will probably alter habitat characteristics, migration patterns, reproduction time, and place of various species2. Such human disturbances may produce new breeding overlaps, breaking the independent evolution of organisms and leading to hybridization (see Glossary, Table 1)3. The role of hybridization in the evolution of several plant and animal taxa has been recognized in the light of newly developed molecular tools4. This has also alerted biologists about the threat this phenomenon may represent to biodiversity when enhanced by anthropogenic factors5. We identified three types of hybridization regarding the reproductive properties of first-generation hybrids (F1). This is proposed as a framework to investigate the demographic and genetic effects of hybridization on biodiversity.
Table 1 Glossary.
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Our perspectives come from the development of modeling simulation approaches applied to various real case studies, which helped us to explore the outcomes of hybridization from both conservation and evolutionary perspectives. We bring here a novel view of conservation guidelines aiming to state the conditions under which hybridization may represent priorities for conservation programmes or, alternatively, new evolutionary opportunities. We highlight that hybridization may certainly lead to biodiversity loss when enhanced by human factors, leading for instance to outbreeding depression or the introgression of maladaptive genes. However, it may also drive the emergence of new biodiversity, reducing the effects of inbreeding depression, and increasing the opportunities to adapt to changing environmental conditions.
Species concept problem and interspecific hybridization
The widely accepted biological species concept formulated by Mayr6 states that species are “groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups”. The key idea under this vision is the reproductive isolation that delimits the species unit. This was already proposed by Georges-Louis Leclerc, Comte de Buffon, more than 260 years ago7. Buffon realized that a horse and a donkey are morphologically more similar than some different races of dogs. However, the reproduction in the first case leads to an infertile offspring (a mule) while in the second case, the offspring is fertile, highlighting that a line can be drawn between organisms that cannot reproduce in order to differentiate species.
Charles Darwin supported a different view and dedicated an entire chapter of “On the Origin of Species” to the hybridization concept8. The observation of interbreeding between distinct morphological species, with different degrees of offspring fertility, from completely sterile to even more fertile than parental species in determined conditions, was an argument against sterility or fecundity as a species delineation factor. Darwin agreed with the notion that species may hardly remain different when free sympatric mating occurs, but supported a more continuous conception of species, influenced by the gradual effect of natural selection. However, the idea of species with various degrees of fertility was abandoned during the modern evolutionary synthesis6,9,10.
Much of the understanding about reproductive isolation and interspecific hybridization has been revealed by experimental studies of Drosophila11. Those works revealed that: (i) reproductive isolation is positively correlated with the phylogenetic distance between hybridizing species; (ii) at the same level of genetic divergence, reproductive isolation is higher between sympatric than allopatric species; and (iii) hybrid offspring follow Haldane’s rule, meaning that if one sex is less viable or sterile, it is more likely to be the heterozygotic sex12,13. During most of the 20th century, interspecific hybridization was considered to be rare in nature, mainly arising by human translocation of species and with a small effect in evolution, with hybrids supposedly having lower fertility in most cases14.
Despite the wide acceptance of reproductive isolation as a key element to define species, a large controversy persists around the biological species concept (Box 1). This is mainly motivated by the semipermeable breeding barriers between some species and the difficulty of testing this notion in organisms with nonoverlapping spatial or temporal distribution ranges15,16.

Box 1 Alternative species concepts

Three of the most popular alternative definitions of species are the ecological, phylogenetic, and evolutionary concepts. Ecological species are of closely related lineage using minimally differentiated adaptive zones, also denominated as ecological niches93. Evolutionary species are defined as ancestral-descendant lineages with their own identity, evolutionary tendency and historical fate94. Phylogenetic species are in turn considered to be the minimal cluster of organisms with a pattern of ancestry and descendance95. These three definitions have also been criticized. The ecological and evolutionary species concepts have been judged to be too vague to determine a cut-off point between species15,17. The phylogenetic species concept has been defended by various authors in the field of conservation biology, who consider it an encompassing view of unique ancestral and derived features for separate species, e.g., refs. 15,19. However, this definition has also been the focus of criticism, mainly due to an inflated number of species16. This is because different regions of the genome may express very different evolutionary histories and because hybridization may also perturb phylogenetic classifications by altering monophyletic lineages20. Mallet96 recognized various cases of speciation that are influenced by fertile hybridization in nature and tried to rescue and adapt the more continuous view of species proposed by Charles Darwin. He understood species as groups of genotypes that remain distinct in the face of actual or potential hybridization96,97. He highlighted the fact that genotypes may remain distinct with reproductive isolation, but this would be a way to maintain species or to reach speciation rather than being a means of species discrimination96. To date, there are around 30 definitions of species and a large debate about species concept and the relation with hybridization, e.g., refs. 15,17,18,98.

Species concept and conservation
A problematic view arises when applying the biological species concept, which does not make room for interspecific hybridization17. The semipermeable barriers between genetically, morphologically or ecologically distinct organisms have motivated a large debate about species and hybridization, e.g., refs. 15,18. This discussion is not superfluous for conservation biology because it delimits the main unit of protection17. Yet, what are the central criteria to delineate the units that deserve protection? Some authors consider that because species are evolutionary units, the most appropriate way to diagnose them objectively is through the phylogenetic species concept19. But the use of the phylogenetic species concept has been criticized because small, isolated populations may become well diagnosed evolutionary lineages through the effect of strong genetic drift, inflating the number of species and rendering protection actions more complex. Other authors have advocated that the criteria to delineate conservation units should rely on evidence of reproductive isolation or reduced reproductive fitness20, but these criteria are less objective and sometimes difficult to evidence.
The debate about species concept and hybridization is not only a matter for biologists, but also for scientists from very different domains, as well as politicians who define legal aspects of wildlife protection21. In this sense, Pasachnik et al.22 propose that whatever else a species is, in the field of conservation biology it should be a group of organisms deserving legal protection because its extinction would constitute a meaningful loss of biodiversity. The evolution of biodiversity represents a continuum, in which speciation processes may occur slowly or relatively fast, but will always have a period of uncertainty regarding genetic differentiation between emerging species23. Conservation biology may therefore consider the level of uncertainty due to hybridization by protecting biodiversity as a dynamic system, which is not focused on reproductive isolation to delimit discrete units, but on the sum of features for which the loss of certain organisms may represent a detrimental effect on biodiversity.
Evolution of new biodiversity
Botanists first highlighted the important role of natural hybridization on the speciation process of several species, i.e., in generating new biodiversity, e.g., 24,25. Later, zoologists recognized the major evolutionary effects of introgression on numerous insects e.g., ref. 26, fishes, e.g. ref. 27, amphibians, e.g., ref. 28, reptiles, e.g., ref. 29, birds, e.g., ref. 30, mammals, e.g., ref. 31; and other organisms, e.g., ref. 32, including modern humans (Box 2). There are around 25% of plants and 10% of animals that are currently known to hybridize with another species and the effect of this phenomenon in evolution is considered to be much more important than previously thought33.
Species can naturally change their historical home range in response to changing environmental conditions and meet closely related taxa34. Several species carry signatures of hybrid ancestry from the last Ice Age period, e.g., ref. 27. For this reason we can find DNA of brown bears in polar bears, because ancient hybridization events occurred during the Pleistocene35. The Bering Land Bridge recurrently emerged during this time, allowing organisms to migrate between Eurasia and North America, leading to opportunities of hybridization, such as those observed between Canada lynx (Lynx canadensis) and Eurasian lynx (Lynx lynx)31. Organisms can have introgressed genes from locally extinct species even if they have never been in contact, because a third species, acting as a temporal bridge to gene flow, has hybridized with both of them e.g., ref. 36.
Natural selection may fix beneficial alleles obtained by hybridization or, to the contrary, remove detrimental introgressed alleles. Adaptive introgression has been important for several speciation processes33. For instance, the antipredatory mimicry of three Heliconius butterflies in South America has been acquired by interspecific hybridization, for which the parts of the genome related to color patterns have more introgressed alleles than other regions of the genome37. Introgressed alleles can rapidly spread among individuals when they are related to adaptive traits. For example, “warfarin” is a rodenticide that was developed in 1948 to control house mice (Mus musculus). Mice started to be resistant during the 1960s by acquiring a single gene from the Algerian mouse (M. spretus) through hybridization38. These species were isolated until the development of human agricultural lands. They rarely interbreed and hybrids have limited survival with half of them being sterile, but the resistance gene rapidly spread across Europe. In Germany, where both species do not mingle, one third of house mice have the introgressed resistance gene coming from Algerian mice38. A similar case was documented between two species of mosquitos that are vectors of malaria and have different levels of resistance to an insecticide39. The insecticide acted as a selective pressure driving the spread of resistant alleles obtained by hybridization, even when hybrids had reduced fertility40. The reduced fertility of the offspring is therefore not necessarily selected against and can also represent adaptive mate choice41.
Opportunities for speciation as a result of hybridization can be generated when hybrids exploit unique ecological niches. For instance, a rapid incipient speciation was recently observed in the offspring of two species of yeast, Saccharomyces paradoxus and S. cerevisiae, whose hybrids have the potential to exploit a unique ecology that is intermediate between those of the parental species32. The new genetic architecture generated by hybridization can thus also facilitate ecological divergence, promoting a speciation process by exploiting a specific niche, e.g., ref. 42.
Positive selection can fix adaptive alleles and purifying selection can remove the detrimental alleles, e.g., ref. 27, but introgressed genes can remain even without the effect of natural selection. Neutral introgressed alleles can persist in high proportion, even when the original species is extinct. Currat et al.43 demonstrated through computer simulations and by a review of the literature, that invasive species in range expansion may carry a large quantity of neutral alleles that are introgressed from a local species. The reverse is not necessarily true unless interbreeding is rare (Fig. 1). When hybridization occurs during the expansion of an invasive species into the territory of a local species, introgression is indeed expected to be much higher in the invasive species than in the local species (Fig. 1). This pattern of asymmetric introgression is generally robust to the density and population structure within both species and to the level of interspecific competition. It results from the hybridization level and from the population demographic imbalance at the wave front of the invasion, in which introgressed alleles that are continuously introduced in the invasive species along its expansion, may surf and reach a higher frequency than expected under a stationary context44. While this pattern may be perturbed by density-dependent dispersal45 and long-distance dispersal46, there are several real cases of asymmetrical introgression between demographically imbalanced species that have been proposed to follow this neutral expectation, e.g. refs. 47,48.
Fig. 1: Expected pattern of introgression of neutral genes between local and invasive organisms in range expansion.

a The context of this expected pattern of introgression is the expansion of an invasive species (in beige) in an area where the local species (in blue) is already in demographic equilibrium. The invasive species starts its colonization from the bottom left side of the area with few individuals. b The level of introgression is asymmetrical and higher in the invasive organisms when the interbreeding rate is large enough (after the dotted line in the x-axis). The value of the admixture rate that delineates this expected higher introgression in the invasive taxon depends on the combination of demographic and migration parameters43. The introgression asymmetry between the two species is due to local alleles continuously introduced at the wave front of the invasive range expansion, with a relatively high probability of increasing in frequency due to the surfing process44. The invasive organisms are not necessarily non-indigenous and may also represent threatened organisms that increase in frequency at the expense of exotic organisms45.

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Box 2 Hybridization and human evolution

Hybridization has probably also played a role in our own evolution when modern humans spread out of Africa and met other closely related hominids. Analyses of ancient DNA estimated around two percent of Neanderthal ancestry in the genome of modern humans outside Africa99,100,101. The introgressed genes may have persisted through neutral processes102 or as a result of positive selection e.g., ref. 103. Recently, it has been proposed that some introgressed alleles, adaptive in the past, may currently be associated with certain diseases104. Modern humans are likely to have met and potentially interbred with other hominids in addition to Neanderthals. Huerta-Sánchez et al105. recognized positive selection in haplotypes related to survival at high altitudes in current Tibetans, which seem to have been introgressed from Denisovans. Other haplotypes from Denisovan ancestry seem to be frequent in the current genome of Melanesians106. Our own genome may thus carry the result of various ancient hybridization events during human evolution107.

Biodiversity loss
Hybridization is considered as a major conservation concern when it is motivated by anthropogenic factors, such as translocation of invasive species or by modification of natural habitats5,49. The breakdown of the reproductive barriers between organisms may disrupt their independent evolution and has already increased the risk of extinction of several plant and animal taxa, e.g., refs. 50,51.
Hybridization may lead to different but potentially interacting mechanisms that threaten species persistence. First, outbreeding depression may represent a significant loss of reproductive value and detonates a rapid extinction when it interacts with a demographic decline. This may be stronger between genetically distant species e.g., ref. 52, but organisms do not need to be distantly related to be affected by outbreeding depression. For instance, the human domestication of Atlantic salmon (Salmo salar) has led to lower fertility when mating with conspecifics in the wild, representing a serious threat for wild salmon in Norway53. Second, native genotypes can disappear by genetic swamping and be replaced by the numerical or competitive advantage of invasive genotypes. Third, the introgression of non-native genes can disrupt local adaptations by introducing maladaptive gene complexes54. Fourth, the behavior of wild animals may be perturbed in a way that is difficult to predict, more particularly when it concerns human domesticated animals55, which have been artificially selected according to human lifestyle and, when spreading their genes in nature, may influence a whole network of ecological interactions, e.g., ref. 56. Fifth, hybridization may affect the effective population size of the interacting species with major consequences for rare or threatened species, which already have a reduced number of breeders57. Finally, an important problem for conservation biology arises when the few remaining individuals of a threatened species show a level of introgression that may cause them to lose their legal protection status when hybrids are not considered to be protected organisms, even though the hybrids may have an ecological function otherwise lost with the extinction of parental species21,58.
The loss of species distinctiveness due to introgression has also been called “speciation reversal”, e.g., ref. 59. This may seriously affect key ecological adaptations that appeared during species radiation. Vonlanthen et al.60 documented the rapid extinction of whitefish (Coregonus spp.) in Swiss lakes, which evolved according to ecological opportunities, but human eutrophication and homogenization of the environment is driving extinction by hybridization and demographic decline. A similar case was documented for cichlid fishes of Lake Victoria (East Africa), for which the coloration pattern is a key character that determines mate choice and reproductive isolation, but the turbidity of the water induced by eutrophication relaxed sexual selection, destroying the diversification mechanism61. Speciation reversal is a conservation concern, because it erodes the ecological and genetic distinctiveness between closely related, but ecologically divergent, species60. In a context of climate change, Owens and Samuk62 refers to hybridization as a double edge sword, because even when increasing the pool of potentially adaptive genes, some of these genes may be related to reproductive isolation, weakening any reproductive barrier. The various cases of hybridization leading speciation reversal, e.g., refs. 59,61, suggest that the extinction risk may be more extensive than previously thought60.
Hybridization between wild and domesticated organisms is a worldwide problem of conservation. For instance, the main current threat for the persistence of European wildcats (Felis silvestris) is the hybridization with domestic cats (Felis catus)63,64. Domestic cats were originally domesticated from a wildcat inhabiting the Near East (Felis lybica), but they are genetically distinct to all current F. lybica subspecies65. There are still some wildcat populations remaining in Europe, e.g., ref. 66, but the complete admixture and the loss of genetic distinctiveness have already been achieved in some countries67. Domestic dogs (Canis familiaris) can hybridize with any kind of wolf-like canids and have already led to conservation issues in various cases50, such as for the gray wolf (Canis lupus) in Europe, e.g., ref. 68 the coyote (Canis latrans) in North America, e.g., ref. 56 or the Ethiopian wolf (Canis simensis) in Africa, e.g. ref. 69. Ellington and Murray56 found that hybridization with domestic dogs was driving changes in the space occupied by coyotes, suggesting consequences at the ecosystem level. A particular threat is the hybridization of domestic dogs with the Ethiopian wolf, which is the world’s most endangered canid, persisting with around 500 individuals in 6 isolated populations69,70. The detrimental effects of hybridization with domesticated organisms is reinforced, because they far outnumber their wild counterparts, e.g., ref. 71, in which the extinction risk can be particularly accelerated when rare species hybridize with more abundant species.
Genetically modified organisms and genetic engineering have generated a large debate on how to regulate the spread of modified genes in nature through hybridization e.g., ref. 72. Genomic alteration for economic purposes may induce higher fertility and resistance to pathogens that make crops or hybrids highly invasive73. The reduced fertility of the first-generation hybrids (F1) is not a barrier for the spread of advantageous alleles74, which are frequently observed in the wild, e.g., ref. 75 with hybrids becoming invasive in various cases76. The ecological release of their natural predators or pathogens conferred by the resistant alleles has been proposed as a factor that is initiating this invasion73. A serious risk has been detected in the single wild population of rice in Costa Rica (Oryza glumaepatula) that hybridizes with invasive commercial rice (O. sativa)77. The concerns are not only related to modified plant crops, but also to animals of economic interest, usually with unpredictable ecological effects, e.g., ref. 78 or to non-target insects, as has been documented for the monarch butterfly Danaus plexippus of North America, e.g., ref. 79.
Types of hybridization
We defined three main types of hybridization that may be used as a framework for the understanding of the ecological and evolutionary consequences of hybridization (Fig. 2). These categories include: (1) distant species hybridization, mostly preventing gene flow because hybrids are infertile (Type 1) or (2) because homologous chromosomes do not recombine (Type 2); and (3) interbreeding between more closely related taxa, in which homologous chromosomes recognize themselves during meiosis, resulting in gene flow and consequent introgression between parental organisms (Type 3) (Box 3).
Fig. 2: Three types of hybridization regarding the reproductive characteristics of first-generation hybrids (F1).

Type 1 represents infertile or inviable hybrids. Type 2 hybrids are fertile but introgression is prevented in further generations due to the generation of gametes without recombination during gametogenesis in hybrid offspring. Type 3 hybrids are fertile and there is recombination during gametogenesis allowing introgression in further generations. Non-human-induced hybridization represents hybrids naturally found in nature, in which evolutionary opportunities arise when hybrids are fertile. Conservation guidelines are proposed for human-induced hybridization, which are motivated by any anthropogenic factor. They represent either a purely demographic or both a demographic and genetic effect on interbreeding taxa. The conservation priorities to avoid biodiversity loss are highlighted in red and basically represent human-induced hybridization that produces demographic decline or ecological disequilibrium. A potential tool to increase genetic diversity is highlighted in green.

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Fig. 3: Identification of the type of hybridization.

Different steps that may be considered to recognize the type of hybridization when there is evidence of interbreeding between taxa (modified from Quilodrán et al.81).

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Type 1: Infertile hybrids, no introgression
The first type of hybridization does not result in introgression, because offspring are inviable or infertile. This type of hybridization represents an extinction risk when the loss of reproductive value enhances a demographic decline for one (or both) parental species. The reasons could be either because small populations interbreed with more abundant populations and therefore waste reproductive efforts, or because additional threats are accumulated, such as a disease. For example, in the case of hybridization between Atlantic salmon (Salmo salar) and brown trout (Salmo trutta), hybridization alone is likely not a threat, but could lead to the extinction of some local salmon populations that are already threatened by a parasitic disease80. This type of hybridization may be considered an evolutionary dead-end.
Type 2: Fertile hybrids, no introgression
The second type of hybridization results in fertile F1 hybrids, but introgression is prevented because their offspring are clonal or hemiclonal, transmitting a single parental genome, also called hybridization with genome exclusion. We recently showed that the extinction of natives and the invasion of exotic organisms might be reached in very few generations81. For instance, in the case of hybridogenesis between Western European water-frogs (Pelophylax species complex)51, the extinction risk is not genetically driven, but determined by the “demographic flow” between parental species and mediated by hybrid offspring. We previously demonstrated that this hybridization is a highway to extinction, which may be underappreciated because it emulates the result of hybridization type 1 (i.e., only displaying F1 hybrid phenotypes)81. Evolutionary opportunities may emerge from these systems by generating self-reproducing polyploid forms82, which are observed in plants but rarely found in animals83.
Type 3: Fertile hybrids, introgression
The third type of hybridization defines interbreeding with gene flow between parental organisms leading to genomic mixing and therefore to introgression. This type of hybridization may result in two different effects on biodiversity, either a genetic and demographic risk of species extinction5, or the opportunity of adaptation and evolution of novel diversity14. For instance, hybrids may replace native species and facilitate biological invasions as in the case of mallard (Anas platyrhynchos), which has been widely translocated, cohabiting with other duck species and threatening them by hybridization84. In another example, however, genes from extinct hominids may still be found in high frequency in current human populations due to old hybridization events43,85. This type of hybridization can also represent a new evolutionary opportunity by increasing genetic diversity and possibilities of adaptation84.

Box 3 Assignment to hybridization type

The three types of hybridization constitute a useful guideline for the understanding of the genetics and/or demographics effects of hybridization on biodiversity. However, to determine one of these types in a specific real system is not always an easy task to achieve, especially when it regards the past evolution of already extinct organisms or when it regards the projection of long-term effects. For instance, infertile hybrids, but with very small introgressions, are observed between Atlantic salmon and brown trout108. A small level of introgression may be ignored, when there is a short-term effect of hybridization producing extinction risk80, but it would not be the case when projecting evolutionary long-term effects, and even more so when concerning range expansions (see Fig. 1), in which case it would be considered as type 3. Moreover, detection of hybridization type 3 with low levels of introgression strongly depends on the amount of genetic markers evaluated4.
Because hybridization type 1 and type 2 are both producing only F1 phenotypes, we recently developed a genetic framework to determine the type of hybridization81 (Fig. 3). Type 3 is easier to recognize due to multiple hybrid phenotypes being present in a population caused by different levels of introgression. If only F1 phenotypes are observed, often with a phenotype intermediate between parental taxa, we recommend defining hybrid fertility by performing controlled breeding experiments. If these experiments are not possible because, for instance, the few remaining individuals of the involved species are threatened, it would be useful to observe their demography and sex ratio. Hybridization type 2 generally produces a very fast demographic decline, and most of the time favors the production of a single sex (namely females). When hybridization type 2 is suspected, it is important to define whether hybrids’ gametes are producing a single (non-alternative) or both (alternative) parental genomes. This may be clarified with a pool of gametes haplotyping test, which will show whether all gametes of an individual have a single allele per gene, in which case hybridization type 2 will be of the non-alternative form. If two alleles are present for some loci, this test reveals hybridization type 2 of the alternative form. In this last case, a single gamete haplotyping method may be implemented to determine the proportion of gametes generated from each of the parental taxa. If those tests result in more than two haplotypes, it would indicate introgression with very low fitness: either type 1 when regarding ecological short-term effects or type 3 when considering evolutionary long-term effects. Details about the pool of gametes and single gametes haplotyping test are presented in Quilodrán et al.81.

Conservation guidelines
Allendorf et al.49 proposed hybridization categories that are widely used to prioritize conservation actions. They considered three categories, but defined differently than ours: (i) sterile hybrids, (ii) widespread introgression, and (iii) complete admixture. Indeed, they ignore the effect of fertile hybrids without introgression (hybridization type 2), which is the category that may induce faster extinctions. In addition, they considered the anthropogenic motivation a sine qua non condition to distinguish the conservation issues of hybridization. We highlight here that hybridization, even when induced by humans, is potentially representing a source of genetic variation that could be useful for conservation purposes.
The classification of Allendorf et al.49 has been employed during the past 20 years, but the wider understanding of hybridization impact brought by more recent studies allows us to propose a novel view of conservation priorities (Fig. 2). Given our classification, the conservation priorities are also found in human-induced hybridization, but this is not the single cut-off to delimit them. Hybridization type 1 is a conservation concern when promoting demographic decline, either because two species with high density-imbalance interbreed or because hybridization amplifies other existing risks80. Hybridization type 2 is always a threat that may precipitate extinction within very few generations81. Hybridization type 3 is also a priority when affecting key ecological interactions, either by enhancing demographic decline or because it changes the behavior of wild individuals84.
Hybridization types 1 and 3 should not represent a priority when they are not triggering demographic decline or the disruption of ecological functions80,84. We suggest that the resources to protect biodiversity may be redirected either to other conservation issues or other threatened organisms. In such conditions, hybridization type 3 may even be used as a conservation tool to increase genetic diversity. However, all of these should be implemented carefully84. The potential fitness loss and the detrimental ecological effects of hybridization have first to be evaluated, and this is often difficult to achieve. In the first case, controlled breeding experiments may help to assess the fertility of hybrids. If this is not possible, monitoring the demography of parental species may help to evaluate a potential fitness loss due to hybridization. A detrimental ecological effect of hybridization is more difficult to evaluate but the behavior of hybrids may provide valuable information. As an example, in Britain, extent hybridization has been registered between Scottish wildcats and domestic cats86, as well as between European polecat and feral ferrets87. While the phenotype of Scottish wildcats has been seriously affected86, the polecat phenotype has been much less affected due to hybridization87. In both cases, the increased genetic diversity may have a positive effect in front of changing environmental conditions, but the impact of hybridization on the behavior of wildcats55, and on the fitness of the polecat population88, deserve more attention before rejecting hybridization as a threat or proposing it as a conservation management tool.
We propose that phylogenetically closer taxa with similar ecological requirements may offer some guidelines for assisted hybridization as a tool in conservation. For instance, assisted hybrization between subspecies of panthers has promoted the recovery of Florida panthers (Puma concolor coryi) by increasing heterozygosity and decreasing inbreeding, resulting in an overall increase of survival and fitness89. Hybridization between different species has also promoted the recovery of American chestnuts (Castanea dentata) through the transfer of pathogen resistance from Chinese chestnuts (C. mollisima)90. In circumstances where organisms are evolutionarily close and share similar ecologies, and when the local species is on the brink of extinction, hybrids may also represent a subject of protection, even when hybridization is caused by anthropogenic factors. An example is the interspecific hybridization between coral reefs, in which the parental species Acropora palmata and A. cervicornis have been in a critical decline over the last decades, but their hybrids (also called A. prolifera) have increased in several locations91. The hybrids have been shown to be as fit or even more fit than the parental species92. While the parental species are legally protected, protecting hybrids represents a legal challenge, which may help to preserve functional ecosystems otherwise lost with the extinction of the parental species91. More

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The lignite mine at Bełchatów, Central Poland (Fig. 1), has recently yielded abundant Miocene remains of several species of branchiopod microcrustaceans. They pertain to the order Anomopoda or water fleas, families Chydoridae and Bosminidae. The main synapomorphy of the anomopods is the ephippium, a structure in which sexual or resting eggs are deposited and that forms when external conditions deteriorate. The Daphniidae include the well-known and speciose genus Daphnia, a highly specialized pelagic component of the freshwater zooplankton. The ephippium fossilizes well and is mostly the only structure that survives as a fossil. The Chydoridae are mostly found in the littoral of lakes and ponds, among water plants. The Bosminidae are pelagic, like Daphnia, but are much smaller (around 0.5 mm in body size) while Daphnia and other daphniids may reach up to 6–7 mm.
Figure 1

(A) View of the Bełchatów Lignite Mine outcrop (photo from the nineties of the twentieth century, when the material was collected). The sedimentary unit with deposits with the cladoceran fossils is to the lower right of the picture (photo: G. Worobiec). (B) Piece of mudstone with cladoceran remains (Collection KRAM-P 225, stored in the W. Szafer Institute of Botany, Polish Academy of Sciences). On the exposed surface leaves of trees and shrubs as well as the water plant Potamogeton (red circle) are seen (photo: A. Pociecha).

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The fossil-bearing deposits belong to a clayey-sandy (I-P) unit considered to be of mid to late Miocene age1,2. Beside by geology, this age is supported by fission track dating, and fossils of different animal (mollusks, fish) and plant (higher plants, water plants and algae) groups1,2,3,4,5,6,7. The fission track ages were 16.5 to 18.1 million years, while the Miocene, also known as the age of mammals, extended from 23 to 5.3 million years BP.
The Branchiopoda (a class of the Crustacea composed ten extant orders) include some of the most ancient extant crustaceans (the Anostraca or fairy shrimp) with several credible fossils8. However, the fossil record is patchy. While the oldest known anostracan-like representatives date back to the Cambrian, fossils of the four extant so-called cladoceran orders, with an estimated 1,000 extant species, is poor9 and somewhat paradoxical. The order Ctenopoda is known from ca 60 extant species and several Mesozoic fossils (possibly representing orders in their own right) but is rare in the most abundant source of fossils, the sediments of late Pleistocene–Holocene lakes10,11. The Anomopoda, in contrast, have to date a limited Mesozoic fossil record, but their subfossils abound in Holocene lake sediments.
The family Chydoridae is represented in Bełchatów by at least four genera: Alona, Acroperus, Camptocercus, and Chydorus. Alona s.l. rivals Daphnia as the most species-rich genus of the order9. All Alona fossils seen so far had two connected head-pores (Fig. 2), placing them near or in the somewhat controversial genus Biapertura. In modern European faunas, and except for Biapertura affinis, three-pored species are dominant, while two-pored ones tend to be typical of warm-climate faunas. The postabdomen, another typical anomopod structure functioning more or less as a ‘tail’, is similar but not identical to that of Biapertura affinis. Furthermore, distinct other species (Fig. 2) have been found as well.
Figure 2

Alona with two head pores from Miocene mudstone from Bełchatów [(A–C), headshields; (B) arrow, labrum; (D, F, G, H) abdomens; (E) shell with abdomen] (photo: A. Pociecha, E. Zawisza).

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Another type of fossils (Fig. 3) shares characters of the genera Acroperus and Camptocercus. Some body parts suggest the presence of Acroperus cf. harpae, others are Campocercus like. One three-dimensional fossil of a first trunk limb (P1) with soft parts preserved shows an IDL (inner distal lobe) with two setae and a moderately well developed claw. In Acroperus, a seta instead of a claw is found here. In Camptocercus, P1 has a big hook here, and our fossils are therefore intermediate between both genera, suggesting the fossil to belong to an extinct ancestral stage. The genus Chydorus is represented by C. sphaericus-like fossils (Fig. 3), but the postabdomen is needed to decide on its taxonomic status.
Figure 3

Acroperus-Camptocercus from Miocene mudstone at Bełchatów [(A) complete animal with shell, headshield, and first antenna; (B) abdomen; (C) shell; (D) claw; (E) shell with abdomen; (F) headshield with soft parts, viz. first and second trunk limbs with IDL setae conserved] (photo: A. Pociecha, E. Zawisza).

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Significantly, Bosminidae in our material were almost as abundant as Alona, while in this family no pre-Pleistocene fossils were previously known at all. Most body parts have been recovered (Fig. 4), but not the postabdomen, which is diagnostic. There is strong variation in the fossils, like in the length of the first antenna (compare Fig. 4B with Fig. 4C,E) and the posterior mucro of the valve. It is impossible to decide whether these antennae belong to several or to a single cyclomorphotic species. However, the distinctly swollen mucros like in Fig. 4A, arrow, are not known in any modern Bosmina, and suggest an extinct taxon.
Figure 4

Bosmina from Miocene mudstone from Bełchatów [(A, D, F) shell; (B) shell with headshield; (C) headshield with first antenna; (E) first antenna)] (photo: A. Pociecha, E. Zawisza).

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Daphniid fossil evidence goes back to the Jurassic/Cretaceous boundary. Fossils are fairly common but only consist of resting eggs (ephippia). Ephippia can tentatively be identified to genus level, which has led to the idea that the two subfamilies of Daphniidae have been around, apparently unchanged, for more than 140 million years9. This is known as the morphological stasis hypothesis. Its factual basis is weak although we accept that true Daphnia ephippia occur in the cretaceous of China, Mongolia and Australia9. Credible ephippia have also been isolated from Eocene age Messel pit-oil shales11. Moina (a water flea related to Daphnia) ephippia have been recovered from the early Miocene Barstow formation12,13,14. In the latter place, fossils are three-dimensional, like in our case in Bełchatów. It should be emphasized, however, that at Bełchatów no daphniid fossils have to date been discovered.
A variety of plant and animal groups, including water plants like Potamogeton have been documented from the Bełchatów site. Many are as beautifully preserved as the cladocerans3,4,5,6,7 and attempts have been made to reconstruct the climatic environment in which they lived. It is suggested to have been warmer than today (average 13.5°–16.5°C, cooling down gradually towards the end of the Miocene). The water was probably slowly flowing, through what may have been a swamp or a group of oxbow lakes from freshwater ecosystems2,15.
That we recovered no daphniid fossils may refine our insight into the nature of the Bełchatów aquatic environment: We favour the idea of a series of shallow oxbow lakes in a climate warmer than todays, with at least locally an abundance of macrophytes. Chydorids thrive in such environments, and the genus Simocephalus among daphniids also prefers such an environment. Simocephalus is quite common and widespread in modern weedy lakes but it has not been found in Bełchatów either. Like Daphnia, Bosminids are truly planktonic and need open water. Therefore, the Bełchatów lakes were probably a patchwork of macrophyte beds and open water.
So where did all the daphniids go? Daphniids are also relatively large species (up to several mm in size), while chydorids and bosminids are typically below a millimeter in size. This suggests size-selective predation on the zooplankton, most probably by fish16. Fish (tench, pike, and unidentified species) were present at Bełchatów4, at least until the middle Miocene. Tench is an omnivore and Pike is zooplanktivorous in its early stages, turning to piscivory in later life. Predation-driven exclusion of these large cladocerans may therefore well have acted in concert with the physical environment.
Predatory exclusion is seldom absolute, especially in a non-tropical climate with a cold season (‘winter’) during which predation is relaxed. This allows the prey to recover. In Bełchatów, we see no such a recovery, so another factor must have intervened. We suggest this may have been linked to water chemistry. Cladocerans, though of worldwide occurrence, are sensitive to water chemistry, more than the copepods, the second main group making up the zooplankton. We are unable to specify the precise nature behind the chemical exclusion of daphniids, but there are many examples of lakes in which Cladocera are chemically depressed. Sometimes, like in Lake Tanganyika (Africa), and the Mallili lakes (Indonesia), Cladocera are excluded altogether17,18.
In conclusion, our Miocene assemblage significantly expand our insights in anomopod evolution. They resemble modern faunas but at the same time show stronger signs of evolution at the species and at the genus level than expected under the morphological stasis hypothesis based on Daphnia ephippia. We also find clear indications of a species diversity that resembles todays’, with morphologies that look familiar at first sight but prove divergent in the details.
Bosminid fossils used to be known from the Pleistocene only, but in the Bełchatów fauna they are remarkably abundant and some of them show an unfamiliar morphology. This is best interpreted as a case of paleo-competitive release. Daphniids normally dominate the pelagic and keep bosminid abundances down. But their absence gives these small crustaceans a chance to prosper and dominate the open waters. More

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Affiliations

US Geological Survey, Santa Cruz, CA, USA
Amy Gartman

Center for Geomicrobiology, Department of Bioscience, Aarhus University, Aarhus, Denmark
Alyssa J. Findlay

Authors
Amy Gartman

Alyssa J. Findlay

Corresponding author
Correspondence to Amy Gartman. More

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